WO2002085286A2 - Procedes permettant d'utiliser la chimiokine d'organes lymphoides secondaires pour moduler des processus physiologiques chez des mammiferes - Google Patents

Procedes permettant d'utiliser la chimiokine d'organes lymphoides secondaires pour moduler des processus physiologiques chez des mammiferes Download PDF

Info

Publication number
WO2002085286A2
WO2002085286A2 PCT/US2002/012196 US0212196W WO02085286A2 WO 2002085286 A2 WO2002085286 A2 WO 2002085286A2 US 0212196 W US0212196 W US 0212196W WO 02085286 A2 WO02085286 A2 WO 02085286A2
Authority
WO
WIPO (PCT)
Prior art keywords
slc
ceus
tumor
cancer
polypeptide
Prior art date
Application number
PCT/US2002/012196
Other languages
English (en)
Other versions
WO2002085286A3 (fr
WO2002085286A8 (fr
Inventor
Steven M. Dubinett
Robert M. Strieter
Sherven Sharma
Raj K. Batra
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU2002256268A priority Critical patent/AU2002256268A1/en
Publication of WO2002085286A2 publication Critical patent/WO2002085286A2/fr
Publication of WO2002085286A8 publication Critical patent/WO2002085286A8/fr
Publication of WO2002085286A3 publication Critical patent/WO2002085286A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods of using secondary lymphoid organ chemokine to modulate mammalian physiological processes including those associated with pathological conditions such as cancer.
  • Chemokines a group of homologous, yet functionally divergent proteins, direcdy mediate leukocyte migration and activation and playa role in regulating angiogenesis (Baggiolini et al, Rev. Immunol, 15: 675—705, 1997). Chemokines also function in maintaining immune homeostasis and secondary lymphoid organ architecture (Jung et al., Curr. Opin. Immunol, 11: 319-325, 1999). Several chemokines are known to have antitumor activity.
  • Tumor rejection has been noted in various murine tumor models in which tumor cells have been modified with chemokines including MlPl ⁇ , RANTES, lymphotactin, TCA3, JE/MCP-1/MCAF, MIP3 ⁇ , MIP3 ⁇ , and IP-10 (Luster et al, J. Exp. Med., 178: 1057-1065, 1993; Bottazzi et al, J. Immunol, 148: 1280-1285,1992; Kellermann et al., J. Immunol.,162: 3859-3864, 1999; Sallusto et al., Eur. J. Immunol, 28: 2760-2769, 1998; Sozzani et al., J.
  • chemokines including MlPl ⁇ , RANTES, lymphotactin, TCA3, JE/MCP-1/MCAF, MIP3 ⁇ , MIP3 ⁇ , and IP-10
  • Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a high endothelial-derived CC chemokine normally expressed in high endothelial venules and in T-cell zones of spleen and lymph node, that strongly attracts naive T cells and DCs (Cyster et al., J. Exp. Med., 189: 447-450, 1999.24; Ogata et al, Blood, 93: 3225-3232, 1999; Chan et al, Blood, 93: 3610-3616, 1999; Hedrick et al, J.
  • SLC Secondary lymphoid tissue chemokine
  • CCR7 is expressed on naive T cells and mature DC
  • CXCR3 is expressed preferentially on Thl cytokine-producing lymphocytes with memory phenotype (Yoshida et al., J. Biol. Chem. 273:7118; Jenh et al., J. Immunol. 162:3765).
  • DCs are uniquely potent APCs involved in the initiation of immune responses (Banchereau et al., Nature (Lond.), 392: 245-252, 1998). Serving as immune system sentinels, DCs are responsible for Ag acquisition in the periphery and subsequent transport to T-cell areas in lymphoid organs where they prime specific immune responses. SLC recruits both naive lymphocytes and antigen stimulated DCs into T-cell zones of secondary lymphoid organs, colocalizing these early immune response constituents and culminating in cognate T-cell activation (Cyster et al, J. Exp. Med., 189: 447-450, 1999.24).
  • the invention disclosed herein provides animal models which faithfully mimic immune mechanisms in cancer by utilizing host animals and cancer cells that have an essentially identical genetic background. These models are used to demonstrate the capacity of SLC to orchestrate effective cell-mediated immune responses to syngeneic cancer cells. In addition, these models can be used to evaluate host cytokine profiles that are associated with SLC modulated immune responses to syngeneic cancer cells. As disclosed herein, the antitumor efficiency of secondary lymphoid organ chemokine was evaluated in a number of syngeneic models including transgenic mice that spontaneously develop tumors. In these transgenic mice, bilateral multifocal pulmonary adenocarcinomas develop in an organ-specific manner. As compared with compared with allogeneic models known in the art, the spontaneous tumors that arise in this transgenic mouse model do not expresses non-self antigens and therefore resemble human cancers.
  • lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor ⁇ was decreased in response to SLC treatment.
  • splenocytes from SLC-treated mice secreted significandy more IFN- ⁇ and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10.
  • significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides methods for the use of SLC in the regulation of tumor immunity and cancer immunotherapy.
  • a typical embodiment of the invention is a method of inhibiting the growth of a spontaneous cancer in a mammal by administering to the mammal an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of the cancer cells.
  • SLC secondary lymphoid tissue chemokine
  • the SLC has the polypeptide sequence shown in SEQ ID NO: 1.
  • SLC polypeptide is typically administered to a mammal sytemically, via intratumoral injection or via intra-lymph node injection.
  • an expression vector having a polynucleotide encoding a SLC polypeptide is administered to the mammal and the SLC polypeptide is produced by a mammalian cell transduced with the SLC expression vector.
  • a related embodiment of the invention is a method of inhibiting the growth of syngeneic cancer cells (most preferably spontaneous cancer cells) in a mammal comprising administering secondary lymphoid tissue chemokine (SLC) to the mammal; wherein the SLC is administered to the mammal by transducing the cells of the mammal with a polynucleotide encoding the SLC shown in SEQ ID NO: 1 such that the transduced cells express the SLC polypeptide in an amount sufficient to inhibit the growth of the cancer cells.
  • the vector is ac ninistered to a mammal systemically, via intratumoral injection or via intra-lymph node injection.
  • Another embodiment of the invention is a method of effecting or modulating cytokine expression (e.g. changing an existing cytokine profile) in a mammal or in a population of cells derived from a mammal by exposing the population of cells to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of syngeneic tumor cells.
  • SLC secondary lymphoid tissue chemokine
  • Another embodiment of the invention is a method of effecting an increase in the expression of Interferon- ⁇ (IFN- ⁇ ) polypeptide and a decrease in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptide in a population of syngeneic mammalian cells including CD8 positive T cells, CD4 positive T cells, Antigen Presenting Cells and tumor cells by exposing the population of cells to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of the tumor cells.
  • SLC secondary lymphoid tissue chemokine
  • the increase in the expression of Interferon- ⁇ (IFN- ⁇ ) polypeptides is at least about two-fold and a decrease in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptides is at least about two-fold as measured by an enzyme linked immunoadsorbent (ELISA) assay.
  • IFN- ⁇ Interferon- ⁇
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • FIGURE 1 SLC mediates antitumor responses in immune competent mice: requirement for CD4 and CD8 lymphocyte subsets.
  • 3LL (H-2d) or L1C2 (H-2b) cells (10 5 ) were inoculated s.c. into the right supra scapular area in C57BL/6 and BALB/c mice.
  • FIGURE 2 Intratumoral SLC administration augments the cytolytic capacity of lymph node (LN)-derived lymphocytes.
  • the cytolytic capacity of lymph node-derived lymphocytes from SLC-treated and diluent control tumor-bearing mice was determined after 1 week of stimulation with irradiated 3LL tumors.
  • Lymph node-derived lymphocytes (5 x 10 6 cells /ml) were cultured with irradiated 3LL (10 5 cells /ml) tumors at a ratio of 50:1 in a total volume of 5 ml. After a 5-day culture, the lymph node-derived lymphocytes cytolytic capacity was assessed against 51 Cr-labeled 3LL tumor targets.
  • FIGURES 3A-3E SLC mediates potent antitumor responses in a murine model of spontaneous lung cancer.
  • the antitumor efficacy of SLC was evaluated in the spontaneous bronchogenic carcinoma model in transgenic mice in which the SN40 large T Ag is expressed under control of the murine Clara cell-specific promoter, CC-10 (Gabrilovich et al., Blood, 92: 4150-4166, 1998). Mice expressing the transgene develop diffuse bilateral bronchoalveolar carcinoma and have an average lifespan of 4 months.
  • mice in all of the treatment groups were sacrificed, and their lungs were isolated and embedded in paraffin. H&E staining of paraffin-embedded lung tumor sections from control-treated mice evidenced large tumor masses throughout both lungs without detectable lymphocytic infiltration (3 A and 3C). In contrast, the SLC therapy group evidenced extensive lymphocytic infiltration with marked reduction in tumor burden (3B and 3D).
  • FIGURES 4A-4B Intratumoral aclministration of Ad-SLC reduces lung cancer growth in vivo. Mice were inoculated with 100,000 L1C2 tumor cells and after 5 days treated intratumorally once a week for three weeks with either 10 8 pfu of Ad-CN or Ad- SLC. At this MOI, of Ad-SLC, L1C2 tumor cells transduced in vitro secreted 10 ng/ml/ 10 6 cells /24 hr of SLC. The reduction in tumor volume over time is shown in graphic form in Figure 4A and the number of mice with complete tumor eradication after therapy is shown in table form in Figure 4B.
  • FIGURES 5A and 5B show Tables IA and IB respectively.
  • Table IA shows Intratumoral SLC administration promotes Thl cytokine and antiangiogenic chemokine release and a decline in immunosuppressive mediators. Cytokine profiles in tumors were determined in mice treated intratumorally with SLC and compared with those in diluent- treated control mice bearing tumors. Non-necrotic tumors were harvested, cut into small pieces, and passed dirough a sieve. Tumors were evaluated for the presence of IL-10, IL-12, GM-CSF, IFN- ⁇ , TGF- ⁇ , NEGF, MIG, and IP-10 by ELISA and for PGE 2 by EIA in the supernatants after overnight culture.
  • mice treated intratumorally with SLC had significant reductions of PGE 2 , NEGF, IL-10, and TGF- ⁇ but an increase in IF ⁇ - ⁇ , GM-CSF, IL-12, MIG, and IP-10. Experiments were repeated twice.
  • Table IB shows how SLC treatment of CC-10 Tag mice promotes Type 1 cytokine and antiangiogenic chemokine release and a decline in the immunosuppressive and angiogenic cytokines TGF- ⁇ and NEGF.
  • CC-10 mice treated with SLC had significant reductions in NEGF and TGF- ⁇ but a significant increase in IF ⁇ - ⁇ , IP-10, IL-12, MIG, and GM-CSF.
  • splenocytes from SLC- treated CC-10 mice had reduced levels of IF ⁇ - ⁇ , IP-10, MIG, and IL-12 but decreased TGF- ⁇ levels as compared with diluent-treated CC-10 mice. Values given reflect mean ⁇ SE for six mice/group.
  • Figures 6A and 6B show Tables 2A and 2B respectively.
  • Table 2A shows that SLC increases the frequency of CD4 and CD 8 lymphocyte subsets secreting IF ⁇ - ⁇ and GM-CSF and CDllc + DEC205-expressing DC.
  • Single-cell suspensions of tumor nodules and lymph nodes from SLC and diluent-treated tumor-bearing mice were prepared. Intracytoplasmic staining for GM-CSF and IF ⁇ - ⁇ and cell surface staining for CD4 and CD8 T lymphocytes were evaluated by flow cytometry. DC that stained positively for cell surface markers CDllc and DEC205 in lymph node and tumor nodule single-cell suspensions were also evaluated.
  • Cells were identified as lymphocytes or DC by gating based on the forward and side scatter profiles: 15,000 gated events were collected and analyzed using Cell Quest software. Within the gated T lymphocyte population, intratumoral injection of SLC led to an increase in the frequency of CD4 and
  • Intracytoplasmic staining for GM-CSF and IF ⁇ - ⁇ and cell surface staining for CD4 and CD8 T lymphocytes were evaluated by flow cytometry. DCs that stained positive for cell surface markers CDllc and DEC205 in lymph node, tumor nodule, and spleen single-cell suspensions were also evaluated. Cells were identified as lymphocytes or DCs by gating based on the forward and side scatter profiles; 15,000 gated events were collected and analyzed using Cell Quest software.
  • Figures 7A and 7B show Tables 3A and 3B respectively.
  • Figure 3A shows the specific systemic induction of type 1 cytokines and down-regulation of IL-10 after SLC treatment.
  • Splenic or lymph node-derived lymphocytes (5 X 10 6 cells /ml) were cultured with irradiated 3LL (10 5 cells/ml) tumors at a ratio of 50:1 in a total volume of 5 ml. After overnight culture, supernatants were harvested, and GM-CSF, IFN- ⁇ , IL-12, and IL10 were determined by ELISA.
  • splenocytes and lymph node-derived cells from SLC-treated mice secreted significantiy enhanced levels of IFN- ⁇ , GM-CSF, and IL-12 but reduced levels of IL-10 compared with diluent-treated bearing mice. Results are expressed as picograms per milliliter. Experiments were repeated twice.
  • Table 3B shows the systemic induction of type 1 cytokines and downregulation of IL-10 after SLC treatment. Splenic lymphocytes (5 X 10 6 cells/ml) were cultured with irradiated CC-10 (10 5 cells/ml) tumors at a ratio of 50:1 in a total volume of 5 ml.
  • Abbreviations used herein include: APC, antigen-presenting cell; SLC, secondary lymphoid organ chemokine; DC, dendritic cell; IP-10, IFN- ⁇ inducible protein 10; TGF- ⁇ , transforming growth factor ⁇ ; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; FBS, fetal bovine serum; mAb, monoclonal antibody; VEGF, vascular endothelial growth factor; EIA, enzyme immunoassay; SV40 TAg, simian virus 40 large T antigen; Ag, antigen; PGE2, prostaglandin E2; PE, phycoerythrin; LN, lymph node.
  • APC antigen-presenting cell
  • SLC secondary lymphoid organ chemokine
  • DC dendritic cell
  • IP-10 IFN- ⁇ inducible protein 10
  • TGF- ⁇ transforming growth factor ⁇
  • GM-CSF
  • the invention is based on the discoveries disclosed herein that Secondary Lymphoid-Tissue Chemokine (SLC) modulates cytokine profiles in an immune response to syngeneic tumor cells and can inhibit the growth of these cells.
  • SLC Secondary Lymphoid-Tissue Chemokine
  • the disclosure provided herein demonstrates the antitumor efficiency of SLC in a clinically relevant mouse model where the mice spontaneously develop tumors.
  • injection of recombinant SLC e.g. in the axillary lymph node region
  • SLC injection led to significant increases in CD4 and CD 8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen.
  • the cellular infiltrates observed at the site of the syngeneic tumors were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN- ⁇ inducible protein 10, and monokine induced by IFN- ⁇ (MIG).
  • lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor ⁇ was decreased in response to SLC treatment.
  • splenocytes from SLC-treated mice secreted significantly more IFN- ⁇ and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10.
  • Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.
  • DCs are potent APCs that function as principle activators of T cells
  • SLC the capacity of SLC to facilitate the colocalization of both DC and T cells is shown to reverse tumor-mediated immune suppression and orchestrate effective cell mediated immune responses in a syngeneic context.
  • SLC has been found to have potent angiostatic effects (Soto et al, Annu.Rev. Immunol, 5: 675-705, 1997), thus adding additional support for its use in cancer therapy.
  • the antitumor activity of SLC is determined in the spontaneous model for lung cancer by injecting recombinant SLC into the axillary lymph node region of the transgenic mice.
  • the rationale for injecting SLC in the lymph node region was to colocalize DC to T-cell areas in the lymph nodes where they can prime specific antitumor immune responses. In many clinical situations access to lymph node sites for injection may also be more readily achievable than intratumoral administration. These results show that this approach is effective in generating systemic antitumor responses.
  • SLC injected in the axillary lymph node regions of the CC-10 TAg mice evidenced potent antitumor responses with reduced tumor burden and a survival benefit as compared with CC-10 TAg mice receiving diluent control injections.
  • the reduced tumor burden in SLC-treated mice was accompanied by extensive lymphocytic as well as DC infiltrates of the tumor sites, lymph nodes, and spleens.
  • the cytokine production from tumor sites, lymph nodes, and spleens of the CC- 10 TAg mice was also altered as a result of SLC therapy.
  • the following cytokines were measured: VEGF, IL-10, PGE-2, TGF- ⁇ , IFN- ⁇ , GMCSF, IL-12, MIG, and IP-10 (Table IB).
  • the production of these cytokines was evaluated for the following reasons: the tumor site has been documented to be an abundant source of PGE-2, VEGF, IL-10, and TGF- ⁇ , and the presence of these molecules at the tumor site has been shown to suppress immune responses (Huang et al, Cancer Res., 58: 1208—1216, 1998; Gabrilovich et al, Nat.
  • VEGF, PGE-2, and TGF- ⁇ have also been documented previously to promote angiogenesis (Fajardo et al, Lab. Investig., 74: 600-608, 1996; Ferrara, N. Breast Cancer Res. Treat, 36: 127-137, 1995; Tsuj ⁇ et al, CeU, 93: 705-716, 1998).
  • Antibodies to VEGF, TGF- ⁇ , PGE-2, and IL-10 have the capacity to suppress tumor growth in in vivo model systems.
  • VEGF has also been shown to interfere with DC maturation (Gabrilovich et al., Nat.
  • Both IL-10 and TGF- ⁇ are immune inhibitory cytokines that may potendy suppress Ag presentation and antagonize CTL generation and macrophage activation (Sharma et al., J. Immunol, 163: 5020-5028, 1999; BeUone et al, Am. J. Pathol, 155: 537-547, 1999). Although at higher pharmacological concentrations IL-10 may cause tumor reduction, physiological concentrations of this cytokine suppress antitumor responses (Sharma et al, J. Immunol, 163: 5020-5028, 1999; Sun et al, Int. J.
  • IP-10 and MIG are CXC chemokines that chemoattract activated T ceUs expressing the CXCR3 chemokine receptor (Loetscher et al, J. Exp. Med., 184:963-969, 1996). Both IP-10 and MIG are known to have potent antitumor and antiangiogenic properties (Luster et al, J. Exp. Med., 178: 1057-1065, 1993; Brunda et al, J. Exp. Med., 178: 1223-1230, 1993; Arenberg et al, J. Exp. Med.,/5 : 981-992, 1996; Sgadari et al, Blood, 89: 2635-2643, 1997).
  • MIG and IP-10 are potent angiostatic factors that are induced by IFN- ⁇ (Arenberg et al, J. Exp. Med.,184: 981-992, 1996; Strieter et al, Biochem. Biophys. Res. Commun., 210: 51-57, 1995; Tannenbaum et al, J. Immunol, 161: 927-932, 1998) and may be responsible in part for the tumor reduction in CC-10 TAg mice after SLC administration.
  • Both MIG and IP-10 are chemotactic for stimulated CXCR3-expressing T lymphocytes that could additionaUy amplify IFN- ⁇ at the tumor site (Father et al., J. Leukoc.Biol, 61: 246-257, 1997).
  • Flow cytometric determinations revealed that both CD4 and CD8 ceUs were responsible for the increased secretion of GM-CSF and IFN- ⁇ in SLC-treated mice.
  • An increase in GM-CSF in SLC-treated mice could enhance DC maturation and antigen presentation (Banchereau et al, Nature (Lond.), 392: 245-252, 1998). Additional studies are necessary to precisely define the host cytokines that are critical to the SLC-mediated antitumor response.
  • Type 1 cytokines The increase in the Type 1 cytokines was not limited to the lung but was evident systemicaUy.
  • SLC treatment of CC-10 TAg transgenic mice led to systemic increases in Type I cytokines and antiangiogenic chemokines.
  • splenocytes from SLC-treated CC-10 TAg mice had an increase in GM-CSF, IL-12, MIG, and IP-10 as compared with diluent-treated CC-10 TAg mice.
  • lymph node-derived ceUs from SLC-treated mice secreted significantiy enhanced levels of IFN- ⁇ , IP-10, MIG, and IL-12.
  • Type 1 cytokines was in part attributable to an increase in specificity against the autologous tumor; when cocultured with irradiated CC-10 TAg tumor ceUs, splenocytes from SLC- treated CC-10 TAg mice secreted significantiy increased amounts of GM-CSF and IFN- ⁇ but reduced levels of IL-10. CeU surface staining of CC-10 ceUs foUowed by flow cytometry did not show detectable levels of MHC class II molecules. Although the tumor did not show MHC class II expression, CD4+ type 1 cytokine production may have occurred because splenic APC were present in the assay.
  • the antitumor properties of SLC may be attributable to its chemotactic capacity in colocalization of DCs and T ceUs, as weU as the induction of key cytokines such as IFN- ⁇ , IP-10, MIG, and IL-12.
  • additional studies can delineate the importance of each of these cytokines in SLC-mediated antitumor responses.
  • the potent antitumor properties demonstrated in this model of spontaneous bronchoalveolar carcinoma provide a strong rationale for additional evaluation of SLC regulation of tumor immunity and its use in immunotherapy for cancers such as cancers of the lung.
  • Typical embodiments include methods of modulating syngeneic physiological processes in mammals, for example effecting an increase in the expression of soluble cytokines such as Interferon- ⁇ (IFN- ⁇ ) polypeptides and a decrease in the expression of soluble cytokines such as Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptides in a population of syngeneic mammaUan ceUs including CD8 positive T ceUs, CD4 positive T ceUs, Antigen Presenting CeUs and tumor ceUs by exposing the population of ceUs to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of the tumor ceUs.
  • a closely related embodiment is a method of treating cancer or hyperproHferative ceU growth in a mammal by administering a therapeuticaUy effective amount of an SLC to the mammal.
  • cancer ceUs are derived from normal host ceUs.
  • the antigenic profile of cancer ceUs closely mimics that of normal ceUs.
  • tumor antigens are not truly foreign and tumor antigens fit more with a self/altered self paradigm, compared to a non-self paradigm for antigens recognized in infectious diseases and organ transplants (see, e.g. Lewis et al, Semin Cancer Biol 6(6): 321-327 (1995)).
  • an important aspect of the present invention is the characterization of the effects of SLC in an animal model where the cancer ceUs are spontaneous and the immune ceUs which respond to the cancer ceUs are therefore syngeneic.
  • syngeneic is known in the art to refer to an extremely close genetic similarity or identity especiaUy with respect to antigens or immunological reactions.
  • Syngeneic systems include for example, models in which organs and ceUs (e.g. cancer ceUs and their non-cancerous counterparts) come from the same individual, and/ or models in which the organs and ceUs come from different individual animals that are of the same inbred strain.
  • Syngeneic models are particularly useful for studying oncogenesis and chemotherapeutic molecules.
  • a specific example of a syngeneic model is the CC-10 TAg transgenic mouse model of spontaneous bronchoalveolar carcinoma described herein.
  • aUogeneic is used to connote a genetic dissimilarity between tissues or ceUs that is sufficient to effect some type of immunological mechanism or response to the different antigens present on the respective tissues or ceUs.
  • aUogeneic model is one in which cancer ceUs from one strain of mice are transplanted into a different strain of mice. AUogeneic models are particularly useful for studying transplantation immunity and for the evaluation of molecules that can suppress the immune response to non-self antigens present on the transplanted tissues.
  • cytokines are crucial mediators of immune response.
  • different cytoldnes, different concentrations of cytokines and/ or different combinations of cytokines are used to generate a specific immune response in a specific context.
  • different immune responses involve different cytokine profiles. Therefore, the inherent differences an immune response to non-self tissues as compared to an immune response to self tissues result in part from inherent differences in the cytokine profiles involved in each response.
  • ceU lines in tissue culture selects for an outgrowth of clones having characteristics associated with the greatest fitness in the culture medium, characteristics which are not necessarily consistent with tumor ceU growth in vivo. Because the CC-10 TAg transgenic mouse model described herein produces spontaneous cancer ceUs (as compared to ceU lines), this model specificaUy avoids the problems associated with the use of ceU lines which have been subjected to specific (and non-specific) selective pressures during their period in tissue culture.
  • the immune system responds to non-self tissues (e.g. aUogeneic transplants) differently than it does to self tissues (e.g. a syngeneic transplant).
  • aUogeneic transplants e.g. a Uogeneic transplants
  • self tissues e.g. a syngeneic transplant.
  • an immune reaction observed in response to a foreign tissue is not predictive of an immune response to a self tissue (that is if an immune response wiU even occur). This is iUustrated, for example, by the need for individuals who have received aUogeneic organ transplants to take immunosuppressive drugs.
  • any clinicaUy relevant model of immune response must take this fundamental aspect of immtinity into account, particularly ones designed to assess an immune response to cancer, a pathology which is characterized by the aberrant growth of self tissues.
  • the transgenic mouse model that is used herein does not expose the animal's immune system to non-self antigens, does not mix ceUs and tissue from strains of mice that have been observed to have different immunological characteristics and is instead directed to evaluating an immune response to spontaneous tumors, the data provided by this model is clinicaUy relevant in the context of human cancers.
  • a number of the methods disclosed herein are related to general methods known in the art that can be used to study the effects of SLC in the context of immunological responses to non-self (i.e. aUogeneic) tissues such as geneticaUy non-identical cancer ceUs transplanted into host animals.
  • the methods disclosed herein may be employed in protocols for treating pathological conditions in mammals such as cancer or immune-related diseases.
  • SLC polypeptide is administered to a mammal, alone or in combination with still other therapeutic agents or techniques. Diagnosis in mammals of the various pathological conditions described herein can be made by the skilled practitioner.
  • Diagnostic techniques are available in the art which aUow, e.g., for the diagnosis ox. detection of cancer ot immune related disease in a mammal.
  • cancers may be identified through techniques, including but not limited to, palpation, blood analysis, x-ray, NMR and the like.
  • diagnostic factors that are known in the art to be associated with cancer may be utilized such as the expression of genes associated with malignancy (including PSA, PSCA, PSM and human glandular kallikrein expression) as weU as gross cytological observations (see e.g. Bocking et al., Anal Quant Cytol 6(2):74-88 (1984); Eptsein, Hum Pathol. 1995 Feb;26(2):223-9 (1995); Thorson et al, Mod Pathol. 1998 Jun;ll(6):543-51; Baisden et al, Am J Surg Pathol. 23(8):918-24 91999)).
  • the methods of the invention are useful in the treatment of hyperproHferative disorders and cancers, and are particularly useful in the treatment of soUd tumors.
  • Types of solid tumors that may be treated according to the methods of the invention include, but are not limited to lung cancer, melanoma, breast cancer, tumors of the head and neck, ovarian cancer, endometrial cancer, urinary tract cancers, stomach cancer, testicular cancer, prostate cancer, bladder cancer, pancreatic cancer, leukemia, lymphoma, bone cancer, Ever cancer, colon cancer, rectal cancer, metastases of the above, and metastases of unknown primary origin.
  • SLC is administered to modulate cytokine profiles and/ or inhibit the growth of spontaneous tumor ceUs of the adenocarcinoma lineage (as is demonstrated herein in the transgenic mouse model).
  • tumor ceUs of the adenocarcinoma lineage can occur spontaneously in a number of different organ systems (see, e.g., Yagi et al, Gan No Rinsho 1984 30(11):1392-1397).
  • Polypeptides useful in the methods of the invention encompass both naturaUy occurring proteins as weU as variations and modified forms thereof.
  • SLC polypeptide or protein is meant a Secondary Lymphoid-Tissue Chemokine.
  • SLC includes naturaUy occurring mammaUan SLCs, and variants and fragments thereof, as defined below.
  • the SLC is of human or mouse origin (see, e.g. SEQ ID NOS: 1 and 2 in Table 4 respectively).
  • the SLC is human SLC.
  • Human SLC has been cloned and sequenced (see, e.g. Nagira et al. (1997) J Biol Chem 272:19518; the contents of which are incorporated by reference). Consequently the cDNA and amino acid sequences of human SLC are known in the art (see, e.g. Accession Nos. BAA21817 and AB002409).
  • Mouse SLC has also been cloned and sequenced (see, e.g.
  • SLC polypeptides for use in the methods disclosed herein can be SLC variants, SLC fragments, analogues, and derivatives.
  • analogues is intended analogues of either SLC or an SLC fragment that comprise a native SLC sequence and structure, having one or more amino acid substitutions, insertions, or deletions.
  • Peptides having one; or more peptoids (peptide mimics) are also encompassed by the term analogues (WO 91/04282).
  • derivatives is intended any suitable modification of SLC, SLC fragments, or their respective analogues, such as glycosylation, phosphorylation, or other addition of foreign moieties (e.g. Pegylation as described below), so long as the desired activity is retained. Methods for masking SLC fragments, analogues, and derivatives are avaUable in the art.
  • a polyol for example, can be conjugated to an SLC molecule at one or more amino acid residues, including lysine residues, as disclosed in WO 93/00109.
  • the polyol employed can be any water-soluble poly(alkylene oxide) polymer and can have a linear or branched chain.
  • Suitable polyols include those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbons.
  • the polyol is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), and thus, for ease of description, the remainder of the discussion relates to an exemplary embodiment wherein the polyol employed is PEG and the process of conjugating the polyol to an SLC protein or variant is termed "pegylation.”
  • PEG poly(ethylene glycol)
  • pegylation the process of conjugating the polyol to an SLC protein or variant
  • other polyols such as, for example, poly(propylene glycol) and polyethylene-polypropylene glycol copolymers, can be employed using the techniques for conjugation described herein for PEG.
  • the degree of pegylation of an SLC variant of the present invention can be adjusted to provide a desirably increased in vivo half-Ufe (hereinafter "half-Hfe"), compared to the corresponding non-pegylated protein.
  • half-Hfe a desirably increased in vivo half-Ufe
  • a variety of methods for pegylating proteins have been described. See, e.g., U.S.
  • Pat. No. 4,179,337 (issued to Davis et al), disclosing the conjugation of a number of hormones and enzymes to PEG and polypropylene glycol to produce physiologicaUy active non-immunogenic compositions.
  • a PEG having at least one terminal hydroxy group is reacted with a coupling agent to form an activated PEG having a terminal reactive group.
  • This reactive group can then react with the ⁇ - and ⁇ -amines of proteins to form a covalent bond.
  • the other end of the PEG molecule can be "blocked" with a non-reactive chemical group, such as a methoxy group, to reduce the formation of PEG-crosslinked complexes of protein molecules.
  • the SLC gene and SLC protein includes the murine and human SLC genes and proteins specificaUy described herein, as weU as biologicaUy active structuraUy and/or functionaUy similar variants or analog of the foregoing.
  • SLC peptide analogs generaUy share at least about 50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLAST criteria). For example, % identity values may be generated by WU-BLAST-2 (Altschul et al, 1996, Methods in Enzymology 266:460-480; http://blast.wusd/ edu/blast/README.ht_ml).
  • SLC nucleotide analogs preferably share 50%, 60%, 70%, 80%, 90% or more nucleic acid homology (using BLAST criteria). In some embodiments, however, lower homology is preferred so as to select preferred residues in view of species-specific codon preferences and/ or optimal peptide epitopes tailored to a particular target population, as is appreciated by those skilled in the art.
  • Fusion proteins that combine parts of different SLC proteins or fragments thereof, as weU as fusion proteins of a SLC protein and a heterologous polypeptide are also included. Such SLC proteins are coUectively referred to as the SLC-related proteins, the proteins of the invention, or SLC.
  • variant refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specificaUy described protein.
  • An analog is an example of a variant protein.
  • the SLC-related gene and SLC-related protein includes the SLC genes and proteins specificaUy described herein, as weU as structuraUy and/ or functionaUy similar variants or analog of the foregoing.
  • SLC peptide analogs generaUy share at least about 50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLAST criteria).
  • SLC nucleotide analogs preferably share 50%, 60%, 70%, 80%, 90% or more nucleic acid homology (using BLAST criteria). In some embodiments, however, lower homology is preferred so as to select preferred residues in view of species-specific codon preferences and/or optimal peptide epitopes taUored to a particular target population, as is appreciated by those skilled in the art.
  • Embodiments of the invention disclosed herein include a wide variety of art- accepted variants or analogs of SLC proteins such as polypeptides having amino acid insertions, deletions and substitutions.
  • SLC variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis Carter et al, Nucl Acids Res., /3. 331 (1986); ZoUer et al, Nucl. Acids Res., 10:6487 (1987)
  • cassette mutagenesis WeUs et al., Gene, 34:315 (1985)
  • restriction selection mutagenesis WeUs et al, Philos. Trans. R. Soc.
  • Such changes typicaUy include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (I) and vice versa.
  • Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V).
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequentiy interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction.
  • preferred scanning amino acids are relatively smaU, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typicaUy a preferred scanning amino acid among this group because it el__minates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant.
  • Alanine is also typicaUy preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol, 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
  • SLC proteins and SLC polypeptide fragments useful in the methods of the present invention must possess SLC biological activity. SpecificaUy, they must possess the desired biological activity of the native protein, that is, the dendritic ceU chemoattractant activity, angiostatic activity or anti-tumor activity as described herein.
  • a "SLC variant'" will exhibit at least 30% of a dendritic ceU-chemoattractant activity, tumor inhibitory activity or angiostatic activity of the SLC. More typicaUy, variants exhibit more than 60% of at least one of these activities; even more typicaUy, variants exhibit more than 80% of at least one of these activities.
  • the biological activity of a SLC protein may also be assessed by examining the ab ⁇ ity of the SLC to modulate cytokine expression in vivo such as effecting an increase in the expression of Interferon- ⁇ (IFN- ⁇ ) polypeptides and a decrease in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptides in a population of syngeneic mammaHan ceUs including CD8 positive T ceUs, CD4 positive T ceUs, Antigen Presenting CeUs and tumor ceUs.
  • IFN- ⁇ Interferon- ⁇
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • the biological activity of a SLC protein may also be assessed by exposing the population of ceUs to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide and examining the abUity that this molecule has to inhibit the growth of syngeneic tumor ceUs.
  • the SLC may be administered directly by introducing a SLC polypeptide, SLC variant or SLC fragment into or onto the subject.
  • the SLC may be produced in situ foUowing the administration of a polynucleotide encoding a SLC polypeptide, SLC variant or SLC fragment may be introduced into the subject.
  • the SLC agents of the invention comprise native SLC polypeptides, native SLC nucleic acid sequences, polypeptide and nucleic acid variants, antibodies, monoclonal antibodies, and other components that are capable of blocking the immune response through manipulation of SLC expression, activity and receptor binding.
  • Such components include, for example, proteins or smaU molecules that interfere with or enhance SLC promoter activity; proteins or smaU molecules that attract transcription regulators; polynucleotides, proteins or smaU molecules that stabilize or degrade SLC mRNA; proteins or smaU molecules that interfere with receptor binding; and the like.
  • any means of suppressing or enhancing SLC activity for example, by interfering with receptor binding, interfering with SLC promoter activity, interfering with gene expression, mRNA stabiHty, or protein stability, etc. can be used to modulate the primary immune response and are encompassed by the invention.
  • the amino acid and DNA sequence of SLC are known in the art. See, for example, Pachynski et al. (1998) J. Immunol. 161:952; Yoshida el al. (1998) J. Biol. Chem.
  • Polynucleotides for use in the methods disclosed herein may be naturaUy occurring, such as aUeUc variants, homologs, orthologs, or may be constructed by recombinant DNA methods or by chemical synthesis.
  • the variant polypeptides may be non-naturaUy occurring and made by techniques known in the art, including mutagenesis.
  • Polynucleotide variants may contain nucleotide substitutions, deletions, inversions and insertions.
  • SLC encoding nucleic acid molecules can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. 5,328,470), implantation or by stereotactic injection (see e.g., Chen et al, PNAS 91:3054-3057 (1994)).
  • Vectors for expression in mammaHan hosts are disclosed in Wu et al.
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene deHvery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more ceUs which produce the gene deHvery system.
  • adenovims vectors and particularly tetracycline-controUed adenovirus vectors. These vectors may be employed to deHver and express a wide variety of genes, including, but not limited to cytokine genes such as those of the interferon gene family and the interleukin gene fam y.
  • a preferred method for deHvery of the expression constructs involves the use of an adenovirus expression vector.
  • adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
  • Adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct in host ceUs with complementary packaging functions and (b) to ultimately express a heterologous gene of interest that has been cloned therein.
  • the expression vector comprises a geneticaUy engineered form of adenovirus.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high liter, wide target-ceU range and high infectivity.
  • Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA repHcation and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA repHcation.
  • the El region (EIA and EIB) encodes proteins responsible for the regulation of transcription of the viral genome and a few ceUular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA repHcation. These proteins are involved in DNA repHcation, late gene expression and host ceU shut-off (Renan, 1990).
  • the products of the late genes are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • MLP major late promoter
  • the MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and aU the mRNAs issued from this promoter possess a S'-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.
  • TPL S'-tripartite leader
  • recombinant adenovirus is generated from homologous recombination between a shuttle vector and a master plasmid which contains the backbone of the adenovirus genome. Due to the possible recombination between the backbone of the adenovirus genome, and the ceUular DNA of the helper ceUs which contain the missing portion of the viral genome, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
  • adenovirus vectors which are repHcation deficient, depend on a unique helper ceU line, designated 293, which was transformed from human embryonic kidney ceUs by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 ceUs, carry foreign DNA in either the El, the E3 or both regions. In nature, adenovirus can package approximately 105% of the wUd-type genome, providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of most adenovirus vectors is at least 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.
  • Helper ceU lines may be derived from human ceUs such as human embryonic kidney ceUs, muscle ceUs, hematopoietic ceUs or other human embryonic mesenchymal or epitheHal ceUs.
  • the helper ceUs may be derived from the ceUs of other mammaHan species that are permissive for human adenovirus.
  • ceUs include, e.g., Vero ceUs or other monkey embryonic mesenchymal or epitheHal ceUs.
  • the preferred helper ceU line is 293.
  • Racher et al (1995) disclosed improved methods for culturing 293 ceUs and propagating adenovirus.
  • natural ceU aggregates are grown by inoculating individual ceUs into 1 Hter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. FoUowing stirring at 40 rpm, the ceU viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as foUows.
  • ceU inoculum resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for I to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated.
  • ceUs are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, foUowing which the volume is increased to 100% and shaking commenced for another 72 h.
  • AdPK adenovirus mediated gene deHvery to multiple ceU types has been found to be much less efficient compared to epitheHal derived ceUs.
  • a new adenovirus, AdPK has been constructed to overcome this inefficiency (Wickham et al., 1996), AdPK contains a heparin-binding domain that targets the virus to heparin-containing ceUular receptors, which are broadly expressed in many ceU types. Therefore, AdPK deHvers genes to multiple ceU types at higher efficiencies than unmodified adenovirus, thus improving gene transfer efficiency and expanding the tissues amenable to efficient adenovirus mediated gene therapy.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional repHcation-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historicaUy been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is repHcation defective and will not have an adenovirus El region.
  • it will be most convenient to introduce the foreign gene expression cassette at the position from which the El-coding sequences have been removed.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in Heu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper ceU line or helper virus complements the E4 defect (Brough et al, 1996).
  • Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 to 10 11 plaque-forming units per ml, and they are highly infective.
  • the Hfe cycle of adenovirus does not require integration into the host ceU genome.
  • the foreign genes deHvered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host ceUs. No severe side effects have been reported in studies of vaccination with wUd-type adenovirus (Couch et al, 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in viva gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1991; Rich et al, 1993).
  • trachea instillation Rosenfeld et al., 1991; 1992
  • muscle injection Rogot et al, 1993
  • peripheral intravenous injections Herz and Gerard, 1993
  • stereotactic inoculation into the brain Le Gal La SaUe et al, 1993.
  • Recombinant adenovirus and adeno-associated virus can both infect and transduce non-dividing human primary ceUs.
  • Adeno-associated virus is also an attractive system for use in construction of vectors for deHvery of and expression of genes as it has a high frequency of integration and it can infect nondividing ceUs, thus making it useful for deHvery of genes into mammaHan ceUs, for example, in tissue culture (Muzyczka, 1992) or in vivo.
  • AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al, 1988; McLaughlin et al., 1988).
  • DetaUs concerning the generation and use of rAAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each incorporated herein by reference.
  • AAV vectors have been used successfuUy for in vitro and in vivo transduction of marker genes (KapHtt et al, 1994; Lebkowski et al, 1988; Samulski et al, 1989; Yoder et al., 1994; Zhou et al, 1994a; Hermonat and Muzyczka, 1984; Tratschin et al, 1985; McLaughlin et al, 1988) and genes involved in human diseases (Flotte et al., 1992; Luo et al, 1994; Ohi et al, 1990; Walsh et al, 1994; Wei et al, 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.
  • AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured ceUs (Muzyczka, 1992).
  • another virus either adenovirus or a member of the herpes virus family
  • helper virus the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991), rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994).
  • recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988: Samulski et al, 1989; each incorporated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example plM4S (McCarty et al, 1991; incorporated herein by reference).
  • ceUs are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function, rAAV virus stocks made in such fashion are contaminated with adenovirus which must be inactivated by heat shock or physicaUy separated from the rAAV particles (for example, by cesium chloride density centrifugation).
  • adenovirus vectors containing the AAV coding regions or ceU lines containing the AAV coding regions and some or aU of the adenovirus helper genes could be used (Yang et al., 1994; Clark et al, 1995).
  • CeU lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al., 1995).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an abiHty to convert their RNA to double-stranded DNA in infected ceUs by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into ceUular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient ceU and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host ceU genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is repHcation-defective.
  • a packaging ceU line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
  • the packaging sequence aUows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988: Temin, 1986; Mann et al, 1983).
  • the media containing the recombinant retroviruses is then coUected, optionaUy concentrated, and used for gene transfer.
  • Retroviral vectors are able to infect a broad variety of ceU types. However, integration and stable expression require the division of host ceUs (Paskind et al, 1975).
  • the restricted host-ceU range and low titer of retroviral vectors can limit their use for stable gene transfer in eukaryotic ceUs.
  • a murine leukemia virus-derived vector has been developed in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus (Burns et al., 1993). These vectors can be concentrated to extremely high titers (109 colony forming units /ml), and can infect ceUs that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. These vectors may facilitate gene therapy model studies and other gene transfer studies that require direct deHvery of vectors in vivo.
  • viral vectors may be employed for construction of expression vectors in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus and herpesviruses may be employed. They offer several attractive features for various mammaHan ceUs (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • the methods of the present invention may be combined with any other methods generaUy employed in the treatment of the particular disease or disorder that the patient exhibits.
  • the methods of the present invention may be used in combination with classical approaches, such as surgery, radiotherapy and the like. So long as a particular therapeutic approach is not known to be detrimental in itself, or counteracts the effectiveness of the SLC therapy, its combination with the present invention is contemplated.
  • any surgical intervention may be practiced in combination with the present invention.
  • any mechanism for inducing DNA damage locaUy within tumor ceUs is contemplated, such as y-irradiation, X-rays, UV-irradiation, microwaves and even electronic emissions and the like.
  • the directed deHvery of radioisotopes to tumor ceUs is also contemplated, and this may be used in connection with a targeting antibody or other targeting means.
  • Cytokine therapy also has proven to be an effective partner for combined therapeutic regimens. Various cytokines may be employed in such combined approaches.
  • cytokines examples include IL-l ⁇ , IL-l ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF- ⁇ , GM-CSF, M-CSF, TNF ⁇ , TNF ⁇ , LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ .
  • Cytokines are administered according to standard regimens, consistent with clinical indications such as the condition of the patient and relative toxicity of the cytokine. Below is an exemplary, but in no way Hmiting, table of cytokine genes contemplated for use in certain embodiments of the present invention.
  • compositions of the present invention can have an effective amount of an engineered virus or ceU for therapeutic adniinistration in combination with an effective amount of a compound (second agent) that is a chemotherapeutic agent as exempHfied below.
  • a compound (second agent) that is a chemotherapeutic agent as exempHfied below.
  • Such compositions will generaUy be dissolved or dispersed in a pharmaceuticaUy acceptable carrier or aqueous medium.
  • chemotherapeutic agents may be used in combination with the therapeutic genes of the present invention. These can be, for example, agents that directiy cross-Hnk DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • chemotherapeutic agents are intended to be of use in the combined treatment methods disclosed herein.
  • Chemotherapeutic agents contemplated as exemplary include, e.g., etoposide (VP- 16), adriamycin, 5-fluorouracU (5FU), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
  • chemotherapeutic agents will be generaUy around those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics.
  • agents such as cisplatin, and other DNA alkylating may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical appHcations of 20 mg/in 2 for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed oraUy and must therefore be deHvered via injection intravenously, subcutaneously, intratumoraUy or intraperitoneaUy.
  • Agents that directly cross-link nucleic acids, specificaUy DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination.
  • Agents such as cisplatin, and other DNA alkylating agents may be used.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyUotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/in2 at 21 day intervals for adriamycin, to 35-50 mg/in2 for etoposide intravenously or double the intravenous dose oraUy.
  • Agents that disrupt the synthesis and fideHty of polynucleotide precursors may also be used. Particularly useful are agents that have undergone extensive testing and are readUy avaUable. As such, agents such as 5-fluorouracU (5-FU) are preferentiaUy used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic ceUs. Although quite toxic, 5-FU, is appHcable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/ day being commonly used.
  • 5-FU is appHcable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/ day being commonly used.
  • Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree, Taxus brevifoHa. It binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Taxol is currendy being evaluated clinicaUy; it has activity against maHgnant melanoma and carcinoma of the ovary. Maximal doses are 30 mg/m 2 per day for 5 days or 210 to 250 mg/m 2 given once every 3 weeks. Of course, aU of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • chemotherapeutic agents that are useful in connection with combined therapy are Hsted in Table B.
  • Each of the agents Hsted therein are exemplary and by no means Hmiting.
  • the skilled artisan is directed to "Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet ster ity, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Hodgkin' s lymphomas primary brain tumors, multiple myeloma, malignant
  • BCNU Carmustine
  • DTIC dacarbazine
  • melanoma Hodgkin' s dimethyltrizenoimida disease, soft- z tissue
  • Fluouracil (5- ovary, head fluorouracil ; and neck,
  • Cytarabine cytosine lymphocytic
  • Vinca Alkaloids Acute lymphocytic leukemia, neuroblastoma,
  • Choriocarcinom a Wilms' tumor, rhabdomyosarco ma, testis,
  • Hodgkin' s lymphomas acute leukemias, breast, genitourinary, thyroid, lung, stomach,
  • Mitomycin (mitomycin bladder, head
  • Testis ovary, bladder, head and neck, lung, thyroid, cervix, endometrium,
  • Cis-DDP Miscellaneo Coordination Cisplatin (cis-DDP) osteogenic us Agents Complexes Carboplatin sarcoma Acute granulocytic leukemia,
  • Mitotane (o.p' -DDD) Adrenal cortex
  • the SLC polypeptides, SLC polypeptide variants, SLC polypeptide fragments, SLC polynucleotides encoding said polypeptides, variants and fragments, and the SLC agents useful in the methods of the invention can be incorporated into pharmaceutical compositions suitable for administration into a mammal.
  • the term "mammal” as used herein refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the mammal is a human.
  • compositions typicaUy comprise at least one SLC polypeptide, SLC polypeptide variant, SLC polypeptide fragment, SLC polynucleotide encoding said polypeptide, variant or fragment, an SLC agent, or a combination thereof, and a pharmaceuticaUy acceptable carrier.
  • pharmaceuticalaUy acceptable carrier is intended to include any and aU solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceuticaUy active substances is weU known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention.
  • Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the, invention is formulated to be compatible with its intended route of administration. The route of administration will vary depending on the desired outcome.
  • injection of the agent at or near the desired site of inflammation or response is utilized.
  • other routes of administration may be warranted depending upon the disease condition. That is, for suppression of neoplastic or tumor growth, injection of the pharmaceutical composition at or near the tumor site is preferred.
  • systemic administration maybe used.
  • routes of systemic administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous appHcation can include the foUowing components: a sterUe diluent such as water for injection, saline solution; fixed oUs, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterUe diluent such as water for injection, saline solution
  • fixed oUs polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • the pharmaceutical composition can be deHvered via slow release formulation or matrix comprising SLC protein or DNA constructs suitable for expression of SLC protein into or around a site within the body.
  • a transient lymph node can be created at a desired implant location to attract dendritic ceUs and T ceUs initiating an immune response.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. That result can be reduction and/ or aUeviation of the signs, symptoms, or causes of a disease or any other desired alteration of a biological system.
  • compositions of the invention comprising SLC polypeptides, SLC polypeptide variants, SLC polypeptide fragments, polynucleotides encoding said SLC polypeptides, variants and fragments, as weU as SLC agents, as defined above, are administered in therapeuticaUy effective amounts.
  • the "therapeuticaUy effective amount” refers to a nontoxic dosage level sufficient to induce a desired biological result. Amounts for administration may vary based upon the desired activity, the diseased state of the mammal being treated, the dosage form, method of administration, patient factors such as age, sex, and severity of disease. It is recognized that a therapeuticaUy effective amount is provided in a broad range of concentrations.
  • an SLC or SLC composition is injected directly into the tumor or into a peritumor site.
  • peritumor site is meant a site less than about 15 cm from an outer edge of the tumor.
  • an SLC or SLC composition is injected into an lymph node that is proximal to the tumor.
  • SLC administration may be to one or more sites. Preferably, SLC administration is at multiple sites within a tumor and/ or surrounding a tumor.
  • the SLC polypeptide is preferably administered to the mammal in a carrier; preferably a pharmaceuticaUy-acceptable carrier.
  • a carrier preferably a pharmaceuticaUy-acceptable carrier.
  • Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences. 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. TypicaUy, an appropriate amount of a pharmaceuticaUy-acceptable salt is used in the formulation to render the formulation isotonic.
  • the carrier include saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably fiom about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of soHd hydrophobic polymers containing, for example, the SLC polypeptide, which matrices are in the form of shaped articles, e.g., films, Hposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of SLC polypeptide being administered.
  • the SLC polypeptide can be administered to the mammal by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal), or by other methods such as infusion that ensure its deHvery to the bloodstream in an effective form.
  • the SLC polypeptide may also be administered by isolated perfusion techniques, such as isolated tissue perfusion, to exert local therapeutic effects. Local or intravenous injection is preferred.
  • Effective dosages and schedules for administering the SLC polypeptides may be determined empirically (e.g. using the models disclosed herein), and making such determinations is within the skiU in the art.
  • Those skilled in the art wiU understand that the dosage of SLC polypeptide that must be administered wiU vary depending on, for example, the mammal which will receive the SLC polypeptide, the route of administration, the particular type of molecule used (e.g. polypeptide, polynucleotide etc.) used and other drugs being administered to the mammal.
  • the SLC polypeptide may be administered sequentiaUy or concurrently with one or more other therapeutic agents.
  • this molecule and therapeutic agent depend, for example, on what type of drugs are used, the pathological condition being treated, and the scheduling and routes of administration but would generaUy be less than if each were used individuaUy. It is contemplated that the antagonist or blocking SLC antibodies may also, be used in therapy. For example, a SLC antibody could be administered to a mammal (such as described above) to block SLC receptor binding.
  • SLC polypeptides of the invention FoUowing administration of a SLC polypeptide to the mammal, the mammal's physiological condition can be monitored in various ways weU known to the skilled practitioner.
  • the therapeutic effects of the SLC polypeptides of the invention can be examined in in vitro assays and using in vivo animal models.
  • a variety of weU known animal models can be used to further understand the role of the SLC in the development and pathogenesis of for instance, immune related disease or cancer, and to test the efficacy of the candidate therapeutic agents.
  • the in vivo nature of such models makes them particularly predictive of responses in human patients.
  • Animal models of immune related diseases include both non-recombinant and recombinant (transgenic) animals.
  • Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing ceUs into syngeneic mice using standard techniques, e.g. subcutaneous injection, tail vein injection, spleen implantation, intraperixoneal implantation, and implantation under the renal capsule.
  • the article of manufacture comprises a container with a label.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition having an active agent which is effective for treating pathological conditions such as cancer.
  • the active agent in the composition is preferably SLC.
  • the label on the container indicates that the composition is used for treating pathological conditions or detecting or purifying SLC, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • the kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a preferred embodiment of the invention is a method of effecting or modulating cytokine expression (e.g. changing an existing cytokine profile) in a mammal or in a population of ceUs derived from a mammal by exposing the population of ceUs to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of syngeneic tumor ceUs such as the spontaneous carcinoma ceUs that arise in the transgenic mouse model described herein.
  • SLC secondary lymphoid tissue chemokine
  • the method described above can be practiced seriaUy as the effects of compounds that have the ability modulate the cytokine profiles is examined.
  • the cytokine profile (and/ or inhibition of tumor growth) in response to SLC in a given cancer model is first examined to determine the effects of SLC in that specific context.
  • the results of such assays can then be compared to the effects that SLC has on a known cancer model such as the transgenic mouse model described herein in order to confirm the effects of SLC in that model.
  • a variation of the method can then be repeated using a test compound in place of SLC and the cytokine profile with the response to the test compound in the model then being examined to identify molecules which can produce physiological effects that are similar or dissimilar to SLC (e.g. modulate cytokine profile and/ or inhibition of tumor growth in a specific way).
  • SLC and a test compound can be added simultaneously to see if the test compound can modulate the effects of SLC in a manner that may have some clinical appHcability, for example to modulate the cytokine profile in a manner that enhances the inhibition of tumor growth, aUows inhibition of growth with fewer side effects etc.
  • the effects of SLC in a given system can be observed or monitored in a number of ways, for example, the effects of SLC can be observed by the evaluation of a change in a cytokine profile, an evaluation the inhibition of tumor growth or tumor killing (e.g. by observing a reduction in tumor size and/ or a reduction in the severity of symptoms associated with the tumor and/ or tumor growth), an increased survival rate (as observed with the transgenic mouse model disclosed herein) and the like.
  • a specific embodiment of this embodiment of the invention is a method of effecting an increase in the expression of Interferon- ⁇ (IFN- ⁇ ) polypeptide and a decrease in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptide in a population of syngeneic mammaHan ceUs including CD8 positive T ceUs, CD4 positive T ceUs, Antigen Presenting CeUs and tumor ceUs comprising exposing the population of ceUs to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of the tumor ceUs and then repeating this method and additionally exposing the population of ceUs to a test compound consisting of a smaU molecule or polypeptide agent. The data from these assays can then be compared to observe effect that the test compound has on the expression of IFN- ⁇ polypeptide or the expression of TGF- ⁇ polypeptide.
  • SLC secondary lymphoid tissue chemokine
  • Any molecule known in the art can be tested for its ability to mimic or modulate (increase or decrease) the activity of SLC as detected by a change in the level of certain cytokines.
  • candidate molecules can be directly provided to a ceU or test subject in vivo or in vitro in order to detect the change in cytokine expression.
  • any lead activator or inhibitor structure known in the art can be used in conjunction with the screening and treatment methods of the invention. Such structures may be used, for example, to assist in the development of activators and/or inhibitors of SLC.
  • This embodiment of the invention is weU suited to screen chemical Hbraries for molecules which modulate, e.g., inhibit, antagonize, or agonize or mimic, the activity of SLC as measured by the change in cytokine levels.
  • the chemical Hbraries can be peptide Hbraries, peptidomimetic Hbraries, chemicaUy synthesized Hbraries, recombinant, e.g., phage display Hbraries, and in vitro translation-based Hbraries, other non-peptide synthetic organic Hbraries, etc.
  • Exemplary Hbraries are commerciaUy avaUable from several sources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In some cases, these chemical Hbraries are generated using combinatorial strategies that encode the identity of each member of the Hbrary on a substrate to which the member compound is attached, thus aUowing direct and immediate identification of a molecule that is an effective modulator. Thus, in many combinatorial approaches, the position on a plate of a compound specifies that compound's composition. Also, in one example, a single plate position may have fiom 1-20 chemicals that can be screened by administration to a weU containing the interactions of interest.
  • Hbraries suitable for use are known in the art and can be used to provide compounds to be tested according to the present invention.
  • Hbraries can be constructed using standard methods. Chemical (synthetic) Hbraries, recombinant expression Hbraries, or polysome-based Hbraries are exemplary types of Hbraries that can be used.
  • the Hbraries can be constrained or semirigid (having some degree of structural rigidity), or linear or nonconstrained.
  • the Hbrary can be a cDNA or genomic expression Hbrary, random peptide expression Hbrary or a chemicaUy synthesized random peptide Hbrary, or non-peptide Hbrary.
  • Expression Hbraries are introduced into the ceUs in which the assay occurs, where the nucleic acids of the Hbrary are expressed to produce their encoded proteins.
  • peptide Hbraries that can be used in the present invention may be Hbraries that are chemicaUy synthesized in vitro. Examples of such Hbraries are given in Houghten et al., 1991, Nature 354:84-86, which describes mixtures of free hexapeptides in which the first and second residues in each peptide were individuaUy and specificaUy defined; Lam et al., 1991, Nature 354:82-84, which describes a "one bead, one peptide" approach in which a soHd phase spHt synthesis scheme produced a Hbrary of peptides in which each bead in the coUection had immobilized thereon a single, random sequence of amino acid residues; Medynski, 1994, Bio/Technology 12:709-710, which describes spHt synthesis and T-bag synthesis methods; and GaUop et al., 1994, J.
  • a combinatorial Hbrary may be prepared for use, according to the methods of Ohlmeyer et al., 1993, Proc. Nati. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Nati. Acad. Sci. USA 91:1614-1618; or Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712.
  • the Hbrary screened is a biological expression Hbrary that is a random peptide phage display Hbrary, where the random peptides are constrained (e.g., by virtue of having disulfide bonding).
  • structuraUy constrained, organic diversity e.g., nonpeptide
  • a benzodiazepine Hbrary see e.g., Bunin et al, 1994, Proc. Nad. Acad. Sci. USA 91:4708-4712) may be used.
  • ConformationaUy constrained Hbraries that can be used include but are not limited to those containing invariant cysteine residues which, in an oxidizing environment, crosslink by disulfide bonds to form cysteines, modified peptides (e.g., incorporating fluorine, metals, isotopic labels, are phosphorylated, etc.), peptides containing one or more non- naturaUy occurring amino acids, non-peptide structures, and peptides containing a significant fraction of (-carboxyglutamic acid.
  • non-peptides e.g., peptide derivatives (for example, that contain one or more non-naturaUy occurring amino acids) can also be used.
  • Peptoids are polymers of non-natural amino acids that have naturaUy occurring side chains attached not to the alpha carbon but to the backbone amino nitrogen. Since peptoids are not easUy degraded by human digestive enzymes, they are advantageously more easUy adaptable to drug use.
  • Hbrary in which the amide functionaHties in peptides have been permethylated to generate a chemicaUy transformed combinatorial Hbrary, is described by Ostresh et al., 1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
  • the members of the peptide Hbraries that can be screened according to the invention are not limited to containing the 20 naturaUy occurring amino acids.
  • chemicaUy synthesized Hbraries and polysome based Hbraries aUow the use of amino acids in addition to the 20 naturaUy occurring amino acids (by their inclusion in the precursor pool of amino acids used in Hbrary production).
  • the Hbrary members contain one or more non-natural or non-classical amino acids or cycHc peptides.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, "-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid; (-Abu, ,-Ahx, 6-amino hexanoic acid; Aib, 2-amino isobutyric acid; 3-amino propionic acid; ornithine; norleucine; norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C"-methyl amino acids, N"-methyl amino acids, fluoro-amino acids and amino acid analogs in general.
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • fragments and/ or analogs of proteins of the invention are screened for activity as competitive or non-competitive inhibitors of activity.
  • combinatorial chemistry can be used to identify modulators.
  • Combinatorial chemistry is capable of creating Hbraries containing hundreds of thousands of compounds, many of which may be structuraUy simUar.
  • WhUe high throughput screening programs are capable of screening these vast Hbraries for affinity for known targets, new approaches have been developed that achieve Hbraries of smaUer dimension but which provide maximum chemical diversity. (See e.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).
  • affinity fingerprinting One method of combinatorial chemistry, affinity fingerprinting, has previously been used to test a discrete Hbrary of smaU molecules for binding affinities for a defined panel of proteins.
  • the fingerprints obtained by the screen are used to predict the affinity of the individual Hbrary members for other proteins or receptors of interest
  • the fingerprints are compared with fingerprints obtained from other compounds known to react with the protein of interest to predict whether the Hbrary compound might simUarly react. For example, rather than testing every Hgand in a large Hbrary for interaction with a complex or protein component, only those Hgands having a fingerprint similar to other compounds known to have that activity could be tested.
  • Kay et al., 1993, Gene 128:59-65 discloses a method of constructing peptide Hbraries that encode peptides of totally random sequence that are longer than those of any prior conventional Hbraries.
  • the Hbraries disclosed in Kay encode totaUy synthetic random peptides of greater than about 20 amino acids in length. Such Hbraries can be advantageously screened to identify complex modulators. (See also U.S. Patent No. 5,498,538 dated March 12, 1996; and PCT PubHcation No. WO 94/18318 dated August 18, 1994).
  • the population of syngeneic mammaHan ceUs used in tiiese methods typicaUy includes CD8 positive T ceUs (i.e. those T ceUs expressing the CD8 antigen), CD4 positive T ceUs (i.e. those T ceUs expressing the CD8 antigen), Antigen Presenting CeUs (APCs) and tumor ceUs.
  • antigen presenting ceU refers to ceUs that constitutively express class II MHC molecules and present stimulatory antigens to T H ceUs.
  • ceUs There are three major classes of ceUs that function as APCs. These classes are macrophages, dendritic ceUs and B lymphocytes. Dendritic ceUs are the most potent among antigen presenting ceUs and are beHeved to be indispensable to the initiation of primary immune responses (see, e.g., Lanzavecchia (1993) Science 260: 937 and Grabbe et al., (1995) Immunology Today 16:117). Tumor ceUs are typicaUy identified through a wide variety of techniques, including but not limited to, palpation, blood analysis, x-ray, NMR and the like.
  • a wide variety of diagnostic factors that are known in the art to be associated with cancer may be utilized to identify a tumor ceUs such as the expression of genes associated with malignancy (e.g. PSA, PSCA, PSM and human glandular kallikrein expression) as weU as gross cytological observations (see e.g. Bocking et al., Anal Quant Cytol. 6(2):74-88 (1984); Eptsein, Hum Pathol. 1995 Feb;26(2):223-9 (1995); Thorson et al., Mod Pathol. 1998 Jun;ll(6):543-51; Baisden et al., Am J Surg Pathol. 23(8):918-24 (1999)).
  • genes associated with malignancy e.g. PSA, PSCA, PSM and human glandular kallikrein expression
  • SLC is administered to modulate cytokine profiles and/ or inhibit the growth of spontaneous tumor ceUs of the adenocarcinoma Hneage as is demonstrated herein.
  • the major forms of lung cancer including adenocarcinoma, squamous ceU carcinoma, smaU ceU carcinoma and large ceU carcinoma represent a continuum of differentiation within a common ceU Hneage and express a number of tumor associated antigens (see, e.g.
  • this method of effecting or modulating cytokine expression entaUs increasing the expression of Interferon- ⁇ (IFN- ⁇ , see, e.g. accession nos. AAB59534 and P01580) polypeptides and/ or decreasing in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ , see, e.g., accession nos. AAA50405 and AAK56116) polypeptides in a population of syngeneic mammaHan ceUs.
  • IFN- ⁇ Interferon- ⁇
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • the increase in the expression of Interferon- ⁇ (IFN- ⁇ ) polypeptides is at least about two-fold and a decrease in the expression of Transforming Growth Factor- ⁇ (TGF- ⁇ ) polypeptides is at least about two-fold as measured by an enzyme Hnked immunoadsorbent (ELISA) assay.
  • ELISA enzyme Hnked immunoadsorbent
  • GM-CSF Granulocyte-Macrophage colony stimulating factor
  • MIG monokine induced by IFN- ⁇
  • IL-12 Interleukin-12
  • NP_ 032377 AAD56385 and AAD56386 IFN- ⁇ inducible protein 10
  • P02778 and AAA02968 polypeptides; as weU as a decrease in Prostaglandin E(2) polypeptides or vascular endotheUal growth factor (VEGF, see, e.g. accession nos. NP_003367 and NP_033531) polypeptides.
  • preferred methods include those that generate a change in the cytokine profiles of these molecules via the administration of SLC. This modulation of polypeptide expression can be determined by any one of the wide variety of methods that are used in the art for evaluating gene expression such as the ELISA assays disclosed herein.
  • the increase and/ or decrease in the expression of the polypeptides is at least about two-fold as measured by an enzyme Hnked immunoadsorbent (ELISA) assay.
  • ELISA enzyme Hnked immunoadsorbent
  • the inhibition of tumor growth can be measured by any one of a wide variety of methods known in the art.
  • the inhibition of the growth of the syngeneic tumor ceUs is measured by quantification of tumor surface area.
  • the syngeneic tumor ceUs are spontaneous cancer ceUs.
  • transgenic which express SN40 large TAg transgene under the control of the murine Clara ceU-specific promoter develop diffuse bUateral bronchoalveolar carcinoma. This model is but one of many syngeneic animal models of cancer known in the art that can be utilized according to the methods described herein (see, also Hakem et al., Annu. Rev. Genet. 2001; 35:209-41; Mundy Semin. Oncol.
  • SLC by a variety of methods, for example by administering SLC polypeptide to a mammal via intratumoral injection, or alternatively administering SLC polypeptide to a mammal via intra-lymph node injection.
  • an expression vector having a polynucleotide encoding a SLC polypeptide is administered to the mammal and the SLC polypeptide is produced by a syngeneic mammaHan ceU that has been transduced with an expression vector encoding the SLC polypeptide.
  • Yet another embodiment of the invention is a method of inhibiting the growth of spontaneous mammaHan cancer ceUs in a population of syngeneic CD8 positive T ceUs, CD4 positive T ceUs and Antigen Presenting CeUs by exposing the population of ceUs to an amount of secondary lymphoid tissue chemokine (SLC) polypeptide sufficient to inhibit the growth of the cancer ceUs.
  • SLC secondary lymphoid tissue chemokine
  • a closely related embodiment of the invention is a method of treating a syngeneic cancer in a mammaHan subject comprising administering a therapeuticaUy effective amount of an SLC to the subject.
  • the SLC is human SLC.
  • the SLC has the polypeptide sequence shown in SEQ ID NO: 1.
  • the SLC polypeptide is administered to a mammal via intratumoral injection, or via intra-lymph node injection.
  • an expression vector having a polynucleotide encoding a SLC polypeptide is administered to the mammal and the SLC polypeptide is produced by a syngeneic mammaHan ceU that has been transduced with an expression vector encoding the SLC polypeptide.
  • the ceUs are exposed to a SLC polypeptide that is expressed by a mammaHan ceU that has been transduced with an expression vector encoding the SLC polypeptide.
  • a related embodiment of the invention consists of syngeneic host ceUs .that have been transduced with an expression vector encoding the SLC polypeptide.
  • the syngeneic host ceUs have been transduced with an expression vector encoding the SLC polypeptide in vivo.
  • Yet another embodiment of the invention is a method of inhibiting the growth of cancer ceUs (most preferably spontaneous cancer ceUs) in a mammal comprising administering secondary lymphoid tissue chemokine (SLC) to the mammal; wherein the SLC is administered to the mammal by transducing the ceUs of the mammal with a polynucleotide encoding the SLC shown in SEQ ID NO: 1 such that the transduced ceUs express the SLC polypeptide in an amount sufficient to inhibit the growth of the cancer ceUs.
  • the vector is atlministered to a mammal via intratumoral injection, or alternatively via intra-lymph node injection.
  • Yet another embodiment of the invention is a method of inhibiting the growth of cancer ceUs (most preferably spontaneous cancer ceUs) in a mammal comprising administering secondary lymphoid tissue chemokine (SLC) ex vivo to the mammaHan ceUs.
  • SLC secondary lymphoid tissue chemokine
  • the SLC is administered to the mammal by transducing the ceUs of the mammal with a polynucleotide encoding the SLC shown in SEQ ID NO: 1 such that the transduced ceUs express the SLC polypeptide in an amount sufficient to inhibit the growth of the cancer ceUs.
  • the SLC is administered as an SLC polypeptide in an amount sufficient to inhibit the growth of the cancer ceUs.
  • the population of ceUs can be removed from the mammal by any one of the variety of methods known in the art.
  • TypicaUy the ceUs are removed from the mammal at a site proximal to the cancer ceUs (e.g. at the site of the tumor or from a lymph node proximal to the tumor) and then reintroduced into the mammal after administration of the SLC (typicaUy a site proximal to the cancer ceUs such as at the site of the tumor or at a lymph node proximal to the tumor).
  • Other embodiments of the invention include methods for the preparation of a medication for the treatment of pathological conditions including cancer by preparing a SLC composition for administration to a mammal having the pathological condition.
  • a related method is the use of an effective amount of a SLC in the preparation of a medicament for the treatment of cancer, wherein the cancer ceUs are syngeneic cancer ceUs.
  • Such methods typicaUy involve the steps of including an amount of SLC sufficient to modulate a cytokine profile as discussed above and/or inhibit the growth of syngeneic (preferably spontaneous) cancer ceUs in vivo and an appropriate amount of a physiologicaUy acceptable carrier.
  • syngeneic (preferably spontaneous) cancer ceUs in vivo
  • a physiologicaUy acceptable carrier As is known in the art, optionaUy other agents can be included in these preparations.
  • EXAMPLE 1 METHODS AND MATERIALS FOR EXAMINING IMMUNOMODULATORY MOLECULES SUCH AS SLC IN SYNGENEIC TRANSPLANTABLE TUMOR MODELS
  • Lewis lung carcinoma (3LL, H-2b), were utilized for assessment of antitumor responses in vivo.
  • the ceUs were routinely cultured as monolayers in 25-cm 3 tissue culture flasks containing RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS (Gemini Bioproducts, Calabasas, CA), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2 mM glutamine (JRH Biosciences, Lenexa, KS) and maintained at 37°C in a humidified atmosphere containing 5% CO z in air.
  • FBS Fermini Bioproducts, Calabasas, CA
  • penicillin 100 U/ml
  • streptomycin 0.1 mg/ml
  • 2 mM glutamine JRH Biosciences, Lenexa, KS
  • the ceU lines were Mycoplasma free, and ceUs were utilized up to the tenth passage before thawing frozen stock ceUs from Hquid N 2 .
  • 105 3LL or L1C2 tumor ceUs were inoculated by s.c. injection in the right suprascapular area of C57BL/6 or BALB/c mice, and tumor volume was monitored three times per week.
  • Five-day-old estabHshed tumors were treated with intratumoral injection of 0.5 ⁇ g of murine recombinant SLC or PBS diluent (Pepro Tech, Rocky Hill, NJ) a ⁇ __ministered three times per week for 2 weeks.
  • the endotoxin level reported by the manufacturer was ⁇ 0.1 ng/ ⁇ g (1 EU/ ⁇ g) of SLC.
  • the amount of SLC (0.5 ⁇ g) used for injection was determined by the in vitro biological activity data provided by the manufacturer. Maximal chemotactic activity of SLC for total murine T ceUs was 100 ng/ml.
  • Tumorigenesis experiments were also performed in which equivalent amounts of murine serum albumin were utilized (Sigma, St. Louis, MO) as an irrelevant protein for control injections. Experiments were also performed in which the SLC was administered at the time of tumor inoculation.
  • tumorigenesis experiments were conducted in SCID beige CB17 mice.
  • SLC was administered s.c. at the time of tumor inoculation and then three times per week.
  • CD4 and CD 8 knockout mice were utilized to determine the contribution of CD4 and CD 8 ceUs in tumor eradication.
  • Two bisecting diameters of each tumor were measured with caHpers. The volume was calculated using the formula (0.4) (ab2), with a as the larger diameter and b as the smaUer diameter.
  • cytokine profiles in tumors, lymph nodes, and spleens were determined in both SLC and diluent-treated mice as previously described (Sharma et al., J. Immunol. 163:5020).
  • Non necrotic tumors were harvested, cut into smaU pieces, and passed through a sieve (BeUco Glass, Nineland, ⁇ J).
  • Tumor-draining lymph nodes and spleens were harvested from SLC-treated tumor-bearing, control tumor-bearing, and normal control mice. Lymph nodes and spleens were teased apart, RBC depleted with double-distilled H20, and brought to tonicity with lx PBS.
  • Tumor nodules were evaluated for the production of IL-10, IL-12, GM-CSF, IF ⁇ - ⁇ , TGF- ⁇ , vascular endotheHal growth factor (VEGF), monokine induced by IF ⁇ - ⁇ (MIG), and IP-10 by ELISA and ⁇ PGE2 by enzyme immunoassay (EIA) in the supernatants after an overnight culture.
  • Tumor-derived cytokine and PGE2 concentrations were corrected for total protein by Bradford assay (Sigma, St. Louis, MO).
  • splenic or lymph node-derived lymphocytes were cocultured with irradiated 3LL (105 ceUs/ml) at a ratio of 50:1 in a total volume of 5 ml. After an overnight culture, supernatants were harvested and GM-CSF, IF ⁇ - ⁇ , IL- 12, and IL-10 determined by ELISA.
  • Cytokine protein concentrations from tumor nodules, lymph nodes and spleens were determined by ELISA as previously described (Huang et al., Cancer Res. 58:1208). Briefly, 96-weU Costar (Cambridge, MA) plates were coated overnight with 4 ⁇ g/ml of the appropriate anti-mouse mAb to the cytokine being measured. The weUs of the plate were blocked with 10% fetal bovine serum (Gemini Bioproducts) in PBS for 30 min. The plate was then incubated with the Ag for 1 h, and excess Ag was washed off with PBS-Tween.
  • the plate was incubated with 2 ⁇ g/ml biotinylated mAb to the appropriate cytokine (PharMingen, San Diego, CA) for 30 min, and excess Ab was washed off with PBS-Tween.
  • the plates were incubated with avidin peroxidase, and after incubation in OPD substrate to the desired extinction, the subsequent change in color was read at 490 nm with a Microplate Reader (Molecular Dynamics, Sunnyvale, CA).
  • the recombinant cytokines used as standards in the assay were obtained from PharMingen.
  • IL-12 Biosource
  • NEGF Oncogene Research Products, Cambridge, MA
  • MIG and IP-10 were quantified by -a modification of a double Hgand method as previously described (Standiford et al., J. Clin. Invest. 86:1945).
  • the MIG and IP-10 Abs and protein were from R&D (MinneapoHs, M ⁇ ).
  • the sensitivities of the IL-10, GM-CSF, IF ⁇ - ⁇ , TGF- ⁇ , MIG, and IP-10 ELISA were 15 pg/ml. For IL-12 and NEGF, the sensitivities were 5 pg/ml.
  • PGE2 concentrations were determined using a kit from Cayman Chemical (Ann Arbor, MI) according to the manufacturer's instructions as previously described (Huang et al., Cancer Res. 58:1208).
  • the EIA plates were read by a Molecular Dynamics Microplate Reader.
  • lymph node-derived lymphocytes (5 x 10 6 ceUs/ml) from SLC-treated and diluent tumor-bearing mice were cultured with irradiated 3LL (10 5 ceUs/ml) tumors at a ratio of 50:1 in a total volume of 5 ml. After a 5-day culture, the lytic capacity of lymph node-derived lymphocytes were determined against chromium-labeled ( 51 Cr, Amersham Arlington, Heights, IL; sp. act.
  • T lymphocytes from single-ceU suspensions of tumor nodules and lymph nodes of SLC- treated and dUuent-treated 3LL tumor-bearing mice were depleted of RBC with distilled, deionized H20 and were evaluated for the presence of intracytoplasmic GM-CSF and IFN- ⁇ .
  • CeU suspensions were treated with the protein transport inhibitor kit GolgiPlug (PharMingen) according to the manufacturer's instructions. CeUs were harvested and washed twice in 2% FBS-PBS.
  • CeUs (5 x 10 5 ) ceUs were resuspended in 200 ⁇ l of 2% FBS-PBS with 0.5 ⁇ g FITC-conjugated mAb specific for ceU surface Ags CD3, CD4, and CD8 for 30 min at 4°C. After two washes in 2% FBS-PBS, ceUs were fixed, permeabilized, and washed using the Cytofix/Cytoperm Kit (PharMingen) foUowing the manufacturer's protocol. The ceU peUet was resuspended in 100 ⁇ l Perm/Wash solution and stained with 0.25 ⁇ g PE-conjugated anti-GM-CSF and anti- IFN- ⁇ mAb for intraceUular staining. CeUs were incubated at room temperature in the dark for 30 min, washed twice, resuspended in 300 ⁇ l PBS, 2% paraformaldeh de solution, and analyzed by flow cytometry.
  • Table 4 below provides iUustrative human and murine SLC polypeptide sequences.
  • EXAMPLE 2 EXAMINING IMMUNOMODULATORY MOLECULES IN SYNGENEIC TRANSPLANTABLE TUMOR MODELS USING SLC AS A ILLUSTRATIVE MOLECULE
  • the disclosure provided herein tests antitumor properties of SLC utilizing two syngeneic transplanted murine lung cancer models.
  • intratumoral SLC administration caused significant reduction in tumor volumes compared with diluent- treated tumor-bearing control mice (p ⁇ 0.01), and 40% of mice showed complete tumor eradication (Figs. 1, A and D).
  • the in vitro proHferation of the tumor ceUs was assessed in the presence of SLC.
  • SLC 200 ng/ml
  • SLC was injected intratumoraUy in tumor-bearing SCID beige CB17 mice. SLC administration did not alter tumor volumes in SCID mice (Fig. IE). Similarly, in CD4 and CD 8 knockout mice, SLC faUed to reduce tumor volumes, indicating that SLC- mediated antitumor responses were both CD4 and CD8 dependent (Fig. 1, B and C).
  • cytokine production from tumor nodules after intratumoral SLC administration was examined.
  • the foUowing cytokines were measured: VEGF, IL-10, PGE2, TGF- ⁇ , IFN- ⁇ , GM-CSF, IL-12, MIG, and IP-10 (Table IA). The production of these cytokines were evaluated for the foUowing reasons.
  • the tumor site has been documented to be an abundant source of PGE-2, VEGF, IL-10, and TGF- ⁇ , and the presence of these molecules at the tumor site have been shown to suppress immune responses (Huang et al., Cancer Res. 58:1208; BeUone et al., Am. J. Pathol. 155:537; Gabrilovich et al., Nat. Med. 2:1096).
  • VEGF, PGE2, and TGF- ⁇ have also previously been documented to promote angiogenesis (Fajardo et al., Lab. Invest. 74:600; Ferrara et al., Breast Cancer Res. Treat. 36:127; 28; TsujH et al., CeU 93:705).
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ TGF- ⁇
  • PGE-2 PGE-2
  • IL-10 TGF- ⁇
  • IL-10 immune inhibitory cytokines that may potently suppress Ag presentation and antagonize CTL generation and macrophage activities, thus enabling the tumor to escape immune detection (Sharma et al., J. Immunol. 163:5020; BeUone et al., Am. J. Pathol. 155:537).
  • mice treated intratumoraUy with SLC had significant reductions of PGE2 (3.5-fold), VEGF (4-fold), IL-10 (2-fold) and TGF- ⁇ (2.3-fold) (Table IA).
  • An overaU decrease in IL-10 and TGF ⁇ at the tumor site after SLC administration may have promoted Ag presentation and CTL generation.
  • the decrease in VEGF and TGF- ⁇ at the tumor site after SLC administration may have contributed to an inhibition of angiogenesis.
  • IFN- ⁇ 5-fold
  • GM-CSF 10-fold
  • IL-12 2- fold
  • MIG 6.6-fold
  • IP-10 (2-fold) after SLC administration Table IA.
  • IFN- ⁇ is a type 1 cytokine that promotes ceU-mediated immunity.
  • Increases in IL-12 (2-fold) could explain the relative increase in IFN- ⁇ (5-fold) at the tumor site of SLC-treated mice (Table IA).
  • the tumor ceUs used for this study do not make detectable levels of IL-12.
  • macrophages and DC are the predominant sources of IL-12 at the tumor site.
  • MIG and IP-10 are potent angiostatic factors that are induced by IFN- ⁇ and may be responsible, in part, for IL-12-mediated tumor reduction (Strieter et al., Biochem. Biophys. Res. Commun. 210:51; Tannenbaum et al., J. Immunol. 161:927; Arenberg et al.,. J. Exp. Med. 184:981).
  • IFN- ⁇ an increase in IFN- ⁇ at the tumor site of SLC-treated mice could explain the relative increase in MIG (6.6-fold) and IP-10 (2-fold) (Table IA).
  • Both MIG and IP-10 are chemotactic for stimulated CXCR3-expressing T lymphocytes, and this could also increase IFN- ⁇ at the tumor site (Farber et al., J. Leukocyte Biol. 61:246).
  • An increase in GM-CSF (10-fold) in the tumor nodules of SLC treated mice could enhance DC maturation and Ag presentation (Banchereau et al., Nature 392:245).
  • SLC did not affect tumor ceU production of VEGF, TGF- ⁇ , IL-10, or PGE-2.
  • lymph node ceUs SLC significantly increased lymph node-derived IL-12 (288 ⁇ 15 pg/ml vs 400 ⁇ 7 pg/ml) whUe decreasing IL-10 (110 ⁇ 5 pg/ml vs 67 ⁇ 1 pg/ml), PGE2 (210 ⁇ 4 pg/ml vs 70 ⁇ 2 pg/ml), and TGF- ⁇ (258 ⁇ 9 pg/ml vs 158 ⁇ 7 pg/ml) production in an overnight in vitro culture.
  • DC are uniquely potent APC involved in the initiation of immune responses, and it is weU documented that SLC strongly attracts mature DC (Chan et al., Blood 93:3610; Banchereau et al., Nature 392:245). Because intratumoral SLC administration led to significant tumor reduction, we questioned whether intratumoral SLC administration led to enhanced DC infiltration of tumor nodules and lymph nodes. Single-ceU suspensions of tumor nodules and lymph nodes from SLC and dUuent-treated tumor-bearing mice were stained for the DC surface markers CDllc and DEC205.
  • lymph node and splenocytes from SLC and dUuent-treated tumor-bearing mice were cocultured with irradiated tumor ceUs for 24 h, and GM-CSF, IFN- ⁇ , IL-10, and IL-12 levels were determined by ELISA.
  • GM-CSF, IFN- ⁇ , IL-10, and IL-12 levels were determined by ELISA.
  • splenocytes and lymph node- derived ceUs from SLC-treated tumor-bearing mice secreted significantiy increased levels of IFN- ⁇ (13- to 28-fold), GM-CSF (3-fold spleen only) and IL-12 (1.3- to 4-fold).
  • EXAMPLE 3 METHODS AND MATERIALS FOR EXAMINING IMMUNOMODULATORY MOLECULES SUCH AS SLC IN SPONTANEOUS TUMOR MODELS 1. CeU Culture.
  • Clara ceU lung tumor ceUs were derived fiom freshly excised lung tumors that were propagated in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS (GeminiBioproducts, Calabasas, CA), penicillin (100 units/ml), streptomycin (O.lmg/ml), and 2 mM of glutamine RH Biosciences, Lenexa, KS) and maintained at 37°C in humidified atmosphere containing 5% C02 in air. After two in vivo passages, CC-10 TAg tumor clones were isolated. The ceU lines were Mycoplasma free, and ceUs were used up to the tenth passage before thawing frozen stock ceUs from Hquid N 2 .
  • the transgenic CC-10 TAg mice in which the SV401arge TAg is expressed under control of the murine Clara ceU-specific promoter, were used in these studies (Magdaleno et al., CeU Growth Differ., 8: 145—155, 1997). AU of the mice expressing the transgene developed diffuse bUateral bronchoalveolar carcinoma. Tumor was evident bUateraUy by microscopic examination as early as 4 weeks of age. After 3months of age, the bronchoalveolar pattern of tumor growth coalesced to form multiple bUateral tumor nodules. The CC-10 TAg transgenic mice had an average Hfe span of 4 months. Extrathoracic metastases were not noted.
  • mice Breeding pairs for these mice were generously provided by Francesco J.De Mayo (Baylor CoUege of Medicine, Houston, TX). Transgenic mice were bred at the West Los Angeles Veteran Affairs vivarium and maintained in the animal research facility. Before each experiment using the CC-10 TAg transgenic mice, presence of the transgene was confirmed by PCR of mouse taU biopsies.
  • the 5' primer sequence was SM19-TAG: 5'-TGGACCTTCTAGGTCTTGAAAGG-3' (SEQ ID NO: 3), and the 3' primer sequence was SM36-TAG: 5'- AGGCATTCCACCACTGCTCCCATT-3' (SEQ ID NO: 4).
  • the size of the resulting PCR fragment is 650 bp.
  • DNA (1 ⁇ g) was ampHfied in a total volume of 50 ⁇ l, which contained 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 200 ⁇ M each deoxynucleotidetriphosphates, 0.1 ⁇ M primers, 2.5 mM MgC12, and 2.5 units of Taq polymerase. PCR was performed in a Perkin-Elmer DNA thermal cycler ( orwalk, CT).
  • the ampHfication profile for the SV40 transgene consisted of40 cycles, with the first cycle denaturation at 94°C for 3 min, annealing at 58°Cfor 1 min, and extension at 72°C for 1 min, foUowed by 39 cycles with denaturation at 94°C for 1 min, and the same annealing and extension conditions.
  • the extension step for the last cycle was 10 min.
  • the products were visualized against molecular weight standards on a 1.5%agarose gel stained with ethidium bromide.
  • AU of the experiments used pathogen- free CC-10 TAg transgenic mice beginning at 4—5 week of age.
  • CC-10 TAg transgenic mice were injected in the axillary node region with murine recombinant SLC (0.5 ⁇ g/injection; Pepro Tech, Rocky HUl, NJ) or normal saline dUuent, which contained equivalent amounts of murine serum albumin (Sigma Chemical Co., St. Louis, MO) as an irrelevant protein for control injections. Beginning at 4-5 weeks of age, SLC or control injections were administered three times per week for 8 weeks. The endotoxin level reported by the manufacturer was ⁇ 0.1 ng/ ⁇ g (1 endotoxin unit/ ⁇ g) of SLC. The dose of SLC (0.5 ⁇ g/injection) was chosen based on our previous studies (Arenberg et al.,. J. Exp.
  • Cytokine Determination from Tumor Nodules, Lymph Nodes, and Spleens The cytokine profiles in tumors, lymph nodes, and spleens were determined in both SLC and dUuent-treated mice as described previously (Sharma et al., J. Immunol., 163: 5020- 5028, 1999). Non-necrotic tumors were harvested and cut into smaU pieces and passed through a sieve (BeUco, Vineland, NJ). Axillary lymph nodes and spleens were harvested from SLC-treated tumor-bearing, control tumor-bearing, and normal control mice.
  • Lymph nodes and spleens were teased apart, RBC depleted with ddH20, and brought to tonicity with 1 x PBS. After a 24-h culture period, tumor nodule supernatants were evaluated for the production of IL-10, IL-12, GM-CSF, IFN- ⁇ , TGF- ⁇ , VEGF, MIG, and IP-10 by ELISA and PGE-2 by EIA. Tumor-derived cytokine and PGE-2 concentrations were corrected for total protein by Bradford assay (Sigma Chemical Co.).
  • splenocytes (5 x 10 6 ceUs/ml), were cocultured with irradiated (100 Gy, Cs 137 x-rays) CC- 10 TAg tumor ceUs (10 5 ceUs/ml) at a ratio of 50:1 in a total volume of 5ml.
  • irradiated 100 Gy, Cs 137 x-rays
  • CC- 10 TAg tumor ceUs (10 5 ceUs/ml) at a ratio of 50:1 in a total volume of 5ml.
  • supernatants were harvested and GM-CSF, IFN- ⁇ , and IL-10 determined by ELISA.
  • Cytokine protein concentrations fiom tumor nodules, lymph nodes, and spleens were determined by ELISA as described previously (Sharma et al., Gene Xhet.,4: 1361—1370, 1997). Briefly, 96-weU Costar (Cambridge, MA) plates were coated overnight with 4 ⁇ g/ml of the appropriate antimouse mAb to the cytokine being measured. The weUs of the plate were blocked with 10% FBS (Gemini Bioproducts) in PBS for 30 min. The plate was then incubated with the antigen for 1 h, and excess antigen was washed off with PBS/Tween 20.
  • the plate was incubated with 2 ⁇ g/ml of biotinylated mAb to the appropriate cytokine (PharMingen) for 30 min, and excess antibody was washed off with PBS/Tween 20.
  • the plates were incubated with avidin peroxidase, and after incubation in O-phenylenediamine substrate to the desired extinction, the subsequent change in color was read at 490 nm with a Molecular Devices Microplate Reader (Sunnyvale, CA).
  • the recombinant cytokines used as standards in the assay were obtained fiom PharMingen.
  • IL-12 Biosource
  • VEGF Oncogene Research Products, Cambridge, MA
  • MIG and IP-10 were quantified using a modification of a double Hgand method as described previously (Standiford et al., J. Clin. Investig., 86: 1945-1953, 1990).
  • the MIG and IP-10 antibodies and protein were obtained fiom R&D (MinneapoHs, MN).
  • the sensitivities of the IL-10, GM-CSF, IFN- ⁇ , TGF- ⁇ , MIG, and IP-10 ELISA were 15 pg/ml.
  • For IL-12 and VEGF the ELISA sensitivities were 5 pg/ml.
  • PGE2 concentrations were determined using a kit from Cayman Chemical Co. (Ann Arbor, MI) according to the manufacturer's instructions as described previously (Huang et al., Cancer Res., 58: 1208-1216, 1998).
  • the EIA plates were read by a Molecular Devices Microplate reader (Sunnyvale, CA).
  • T lymphocytes from single ceU suspensions of tumor nodules, lymph nodes, and spleens of SLC-treated and diluent treated CC-10 TAg transgenic mice were depleted of RBC with distilled, deionized H2O and were evaluated for the presence of intracytoplasmic GM-CSF and IFN- ⁇ CeU suspensions were treated with the protein transport inhibitor kit Golgi Plug (PharMingen) according to the manufacturer's instructions. CeUs were harvested and washed twice in 2% FBS/PBS.
  • CeUs (5 x 10 5 ) were resuspended in 200 ⁇ l of 2% FBS/PBS with 0.5 ⁇ g of FITC-conjugated mAb specific for ceU surface antigens CD3, CD4, and CD8 for 30 min at 4°C. After two washes in 2% FBS/PBS, ceUs were fixed, permeabilized, and washed using the Cytofix/Cytoperm kit (PharMingen) foUowing the manufacturer's protocol.
  • ceU peUet was resuspended in 100 ⁇ l of Perm/Wash solution and stained with 0.25 ⁇ g of PE-conjugated anti-GM-CSF and anti- IFN- ⁇ mAb for intraceUular staining. CeUs were incubated at room temperature in the dark for 30 min and washed twice, resuspended in 300 ⁇ l of PBS/2% paraformaldehyde solution, and analyzed by flow cytometry.
  • EXAMPLE 4 SLC MEDIATES POTENT ANTITUMOR RESPONSES IN A MURINE MODEL OF SPONTANEOUS BRONCHOALVEOLAR CARCINOMA.
  • Example 3 the antitumor efficacy of SLC in a spontaneous bronchoalveolar ceU carcinoma model in transgenic mice in which the SN40 large TAg is expressed under control of the murine Clara ceU-specific promoter,CC-10 was evaluated. (Magdaleno et al., CeU Growth Differ., 8: 145—155, 1997). Mice expressing the transgene develop diffuse bilateral bronchoalveolar carcinoma and have an average Hfe span of 4 months. SLC (0.5 ⁇ g/injection) or the same concentration of murine serum albumin was injected in the axillary lymph node region beginning at 4 weeks of age, three times per week and continuing for 8 weeks.
  • mice were sacrificed in aU of the treatment groups, and lungs were isolated and paraffin embedded. H&E staining of paraffin-embedded lung tumor sections fiom control-treated mice revealed large tumor masses throughout both lungs with minimal lymphocytic infiltration (Fig. 3 A and C). In contrast, SLC-treated mice had significantiy smaUer tumor nodules with extensive lymphocytic infiltration (Fig. 3, B and D). Mice treated with SLC had a marked reduction in pulmonary tumor burden as compared with diluent treated control mice (Fig. 3E). SLC-treated mice had prolonged survival compared with mice receiving control injections. Median survival was 18 ⁇ 2 weeks for control-treated mice, whereas mice treated with SLC had a median survival of 34 ⁇ 3 weeks (P ⁇ 0.001).
  • EXAMPLE 5 SLC TREATMENT OF CC-10 TAG MICE PROMOTES TYPE 1 CYTOKINE AND ANTIANGIOGENIC CHEMOKINE RELEASE AND A DECLINE IN THE IMMUNOSUPPRESSIVE CYTOKINES TGF- ⁇ AND VEGF.
  • Lungs, lymph node, and spleens were harvested, and after a 24-h culture period, supernatants were evaluated for the presence of VEGF, IL-10, IFN- ⁇ , GM-CSF, IL-12, MIG, IP-10, and
  • TGF- ⁇ by ELISA and for PGE-2 by EIA were significantly reduceds in VEGF (3.5-fold) and TGF- ⁇ (1.83-fold) but an increase in IFN- ⁇ (160.5-fold), IP-10 (1.7-fold), IL-12 (2.1-fold), MIG (2.1-fold),and GM-CSF (8.3-fold; Table IB).
  • splenocytes fiom SLC-treated CC-10 TAg mice revealed reduced levels of PGE-2 (14.6-fold) and VEGF (20.5-fold) but an increase in GM-CSF (2.4-fold), IL-12 (2-fold), MIG (3.4-fold), and IP-10 (4.1-fold; Table B).
  • lymph node-derived ceUs-from SLC treated mice secreted significantly enhanced levels of IFN- ⁇ (2.2-fold), IP-10 (2.3-fold), MIG (2.3-fold), and IL-12 (2.5-fold) but decreased levels of TGF- ⁇ (1.8-fold; Table IB).
  • the immunosuppressive mediators PGE-2 and IL-10 were not altered at the tumor sites of SLC-treated mice; however, there was a significant reduction in the level of PGE-2 in the spleen of SLC-treated mice.
  • SLC administration induced significant specific systemic immune responses splenocytes from SLC and dUuent treated CC-10 TAg mice were cocultured in vitro with irradiated CC-10 TAg tumor ceUs for 24 h, and GM-CSF, IFN- ⁇ , and IL-10 were determined by ELISA.
  • splenocytes from SLC-treated tumor-bearing mice secreted significantly increased levels of IFN- ⁇ (5.9-fold) and GM-CSF (2.2-fold). In contrast, IL-10 secretion was reduced 5-fold (Table 3B).
  • EXAMPLE 6 SLC TREATMENT OF CC-10 TAG MICE LEADS TO ENHANCED DC AND T-CELL INFILTRATIONS OF TUMOR SITES, LYMPH NODES, AND SPLEEN.
  • SLC-mediated anti-tumor response is accompanied by the enhanced elaboration of IFN- ⁇ , IP-10 and MIG at the tumor site.
  • IP-10, MIG and IFN- ⁇ are known to have potent anti-tumor activities in vivo.
  • a study was undertaken to determine if the augmentation of these cytokines served as effector molecules in SLC mediated tumor reduction.
  • SLC- mediated anti-tumor responses require the cytokines IP-10, MIG and IFN- ⁇ .
  • IP-10 IFN- ⁇ inducible protein IP-10
  • MIG monokine-induced by IFN- ⁇
  • CeU culture and tumorigenesis model A weakly immunogenic lung cancer, Lewis lung carcinoma (3 LL, H-2 b ) was utilized for assessment of cytokines important for SLC- mediated anti-tumor responses in vivo.
  • the ceUs were routinely cultured as monolayers in 25 cm 3 tissue culture flasks containing RPMI 1640 medium (Irvine Scientific, Santa Anna, CA) supplemented with 10% fetal bovine serum (FBS) (Gemini Bioproducts, Calabasas, CA), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2mM glutamine (JRH Biosciences, Lenexa, KS) and maintained at 37° C in humidified atmosphere containing 5% C0 2 in air.
  • FBS fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 0.1 mg/ml
  • 2mM glutamine JRH Biosciences, Lenexa, KS
  • ceU lines were mycoplasma free and ceUs were utilized up to the tenth passage before thawing frozen stock ceUs from Hquid N 2 .
  • 10 5 3LL tumor ceUs were inoculated by s.c. injection in the right supra scapular area of C57B1/6 and tumor volume was monitored 3 times per week.
  • Five day estabHshed tumors were treated with intratumoral injection of 0.5 ⁇ g of murine recombinant SLC or PBS diluent (Pepro Tech, Rocky HUl, NJ) administered three times per week for two weeks.
  • the endotoxin level reported by the manufacturer was less than O.lng per ⁇ g (lEU/ ⁇ g) of SLC.
  • the amount of SLC (0.5 ⁇ g) used for injection was determined by the in vitro biological activity data provided by the manufacturer. Maximal chemotactic activity of SLC for total murine T ceUs was found to be 100 ng/ml. For in vivo evaluation of SLC-mediated anti-tumor properties we utilized 5 fold more than this amount for each intratumoral injection. Tumorigenesis experiments were also performed in which equivalent amounts of murine serum albumin were utilized (Sigma, St. Louis, Mo) as an irrelevant protein for control injections. 24 hours prior to SLC treatment, and then three times a week, mice were treated i.p.
  • MIG, IP-10 and IFN- ⁇ were quantified using a modification of a double Hgand method as previously described.
  • the MIG and IP10 antibodies and recombinant cytokine proteins were from R&D (MinneapoHs, MN).
  • the IFN- ⁇ antibodies pairs and recombinant cytokine were from Pharmingen.
  • the sensitivities of the IFN ⁇ , MIG and IP-10 ELISA were 15 pg/ml.
  • IFN- ⁇ mediates a range of biological effects that facilitate anticancer immunity.
  • MIG and IP-10 are potent angiostatic factors that are induced by IFN- ⁇ and hence we postulated that in addition to IFN- ⁇ they are be responsible in part for the tumor reduction foUowing SLC administration.
  • IFN- ⁇ at the tumor site of SLC-treated mice could explain the relative increases in IP-10 and MIG.
  • IFN- ⁇ production at the tumor site was found to be dependent on MIG and IP-10 because neutralization of these cytokines caused a decrease in IFN- ⁇ .
  • IFN- ⁇ , MIG and IP-10 in SLC treated mice showed an interdependence since in vivo neutralization of any one of these cytokines caused a concomitant decrease in aU three cytokines.
  • Both MIG and IP-10 are chemotactic for stimulated CXCR3-expressing activated T lymphocytes that could further ampHfy IFN- ⁇ at the tumor site.
  • mice were treated intratumoraUy with 0.5 ⁇ g of recombinant murine SLC three times per week.
  • mice were given the respective cytokine antibody by i.p. injection. The antibodies were administered three times per week.
  • EXAMPLE 8 SLC-MEDIATED ANTI-TUMOR RESPONSES IN A MURINE MODEL OF A GENE THERAPY-BASED APPROACH
  • the pJM17 plasmid that contains the entire El -deleted Ad-5 genome was used as the recombination vector (for illustrative methods see, e.g., Cancer Gene Ther 1997 Jan- Feb;4(l): 17-25).
  • Murine AdSLC was prepared through an in vitro recombination event in 293 ceUs through a recombination event between the shuttle plasmid pMH4 containing the murine SLC cDNA and the pJM17 plasmid.
  • Clones of Ad SLC were obtained by Hmiting dUution analysis of the ability of media to induce cytopathic effect on 293 ceUs and confirmed by murine SLC specific ELISA that we developed in our laboratory.
  • Viral stocks were obtained by ampHfication of the 293 ceUs foUowed by CsCl purification, dialysis and storage as a glycerol (10% vol/vol) stock at -80 °C (see, e.g., Cancer Gene Ther 1997 Jan-Feb;4(l): 17-25).
  • 10 5 ceUs were injected in the right supra scapular region of the mice and 5 days later, the tumors treated with an intratumoral injection of Ad-SLC or control Ad vector once a week for three weeks at pfu's ranging from 10 7 -10 9 .
  • the virus was injected into d e tumor using an insulin syringe with the injectate was deHvered slowly to aUow for an even distribution of the virus particles in the tumor.
  • the in vivo gene transfer methods disclosed herein provide clinicaUy relevant models for treating cancers.
  • these in vivo models are directiy relevant cancer models because the cancer arise in a spontaneous manner (and are therefore syngeneic).
  • the gene therapy methods disclosed herein directiy paraUel the clinical model, that is the administration of a polynucleotide encoding SLC polypeptide.
  • the fact that the administration of this gene therapy vector is shown to reduce tumor burden provides direct evidence which strongly supports the use of such vectors in clinical methods for treating cancer. Consequently this model provides a particularly useful tool for optimizing and characterizing SLC based gene therapies.
  • a human gene therapy-based anti-tumor approach can be employed using a vector such as an adenoviral construct that expresses human SLC cDNA.
  • a vector such as an adenoviral construct that expresses human SLC cDNA.
  • the cDNA for human secondary lymphoid chemokine can be cloned downstream of a promoter that aUows an appropriate degree of expression such as a CMV promoter. .
  • a plasmid such as the pJM17 plasmid that contains the entire El -deleted Ad-5 genome can be used as the recombination vector (for iUustrative methods see, e.g.,
  • Human AdSLC can be prepared through an in vitro recombination event in 293 ceUs through a recombination event between a shuttle plasmid containing the human SLC cDNA and the recombination plasmid.
  • Clones of Ad SLC can be obtained by limiting dUution analysis of the ability of media to induce cytopathic effect on ceUs such as 293 ceUs and confirmed by human SLC specific ELISA that we developed in our laboratory.
  • Viral stocks can be obtained by ampHfication of the ceUs foUowed by CsCl purification, dialysis and storage as a glycerol (10% vol/vol) stock at -80 °C (see, e.g., Cancer Gene Ther 1997 Jan- Feb;4(l):17-25).
  • L1C2 Line 1 alveolar carcinoma ceUs
  • 3LL Lewis Lung carcinoma ceUs
  • 10 5 ceUs can be injected in a region proximal to the tumor and 5 days later, the tumors can be treated with an intratumoral injection of SLC vector once a week for three weeks at pfu's ranging from 10 7 -10 9 .
  • the virus can be injected into the tumor using an insulin syringe with the injectate can be deHvered slowly to aUow for an even distribution of the virus particles in the tumor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne l'aptitude de la chimiokine d'organes lymphoïdes secondaires à inhiber in vivo la croissance de tumeurs syngéniques. L'invention concerne également un traitement du cancer chez un mammifère par administration d'une quantité suffisante d'une chimiokine d'organe lymphoïde secondaire. Les chimiokines d'organes lymphoïdes secondaires convenant pour le traitement selon l'invention sont essentiellement des polypeptides, des variantes et des fragments de chimiokines d'organes lymphoïdes secondaires, et des acides nucléiques correspondants.
PCT/US2002/012196 2001-04-18 2002-04-18 Procedes permettant d'utiliser la chimiokine d'organes lymphoides secondaires pour moduler des processus physiologiques chez des mammiferes WO2002085286A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002256268A AU2002256268A1 (en) 2001-04-18 2002-04-18 Methods of using secondary lymphoid organ chemokine to modulate physiological processes in mammals

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28484501P 2001-04-18 2001-04-18
US60/284,845 2001-04-18

Publications (3)

Publication Number Publication Date
WO2002085286A2 true WO2002085286A2 (fr) 2002-10-31
WO2002085286A8 WO2002085286A8 (fr) 2003-04-03
WO2002085286A3 WO2002085286A3 (fr) 2004-04-01

Family

ID=23091745

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/012196 WO2002085286A2 (fr) 2001-04-18 2002-04-18 Procedes permettant d'utiliser la chimiokine d'organes lymphoides secondaires pour moduler des processus physiologiques chez des mammiferes

Country Status (3)

Country Link
US (1) US20030175801A1 (fr)
AU (1) AU2002256268A1 (fr)
WO (1) WO2002085286A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019057808A1 (fr) 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050281782A1 (en) * 2004-06-21 2005-12-22 Howard Kaufman Novel recombinant poxvirus composition and uses thereof
JP7305300B2 (ja) * 2014-11-05 2023-07-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 併用免疫療法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006169A1 (fr) * 1994-08-23 1996-02-29 Human Genome Sciences, Inc. Chemokine beta 9 humaine
US5767097A (en) * 1996-01-23 1998-06-16 Icn Pharmaceuticals, Inc. Specific modulation of Th1/Th2 cytokine expression by ribavirin in activated T-lymphocytes
US5871723A (en) * 1995-06-06 1999-02-16 The Regent Of The University Of Michigan CXC chemokines as regulators of angiogenesis
US6403370B1 (en) * 1997-02-10 2002-06-11 Genstar Therapeutics Corporation Oncolytic/immunogenic complementary-adenoviral vector system

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006169A1 (fr) * 1994-08-23 1996-02-29 Human Genome Sciences, Inc. Chemokine beta 9 humaine
US5871723A (en) * 1995-06-06 1999-02-16 The Regent Of The University Of Michigan CXC chemokines as regulators of angiogenesis
US5767097A (en) * 1996-01-23 1998-06-16 Icn Pharmaceuticals, Inc. Specific modulation of Th1/Th2 cytokine expression by ribavirin in activated T-lymphocytes
US6403370B1 (en) * 1997-02-10 2002-06-11 Genstar Therapeutics Corporation Oncolytic/immunogenic complementary-adenoviral vector system

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOTECHDS [Online] NANDA ET AL.: 'Induction of anti-self-immunity to cure cancer: meeting review; gene therapy, adoptive immunotherapy and antitumor recombinant vaccine production (conference report)', XP002972856 Retrieved from STN Database accession no. 1995:12009 & CELL vol. 82, 1995, pages 13 - 17 *
DILLOO ET AL.: 'Combined chemokine and cytokine gene transfer enhances antitumor immunity' NATURE MEDICINE vol. 2, October 1996, pages 1090 - 1095, XP002119425 *
HEDRICK ET AL.: 'Identification and characterization of a novel beta chemokine containing six conserved cysteines' THE JOURNAL OF IMMUNOLOGY vol. 159, August 1997, pages 1589 - 1593, XP002073210 *
NAGIRA ET AL.: 'Molecular cloning of a novel human CC chemokine secondary lymphoid-tissue chemokine that is a potent chemoattractant for lymphocytes and mapped to chromosome 9p13' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 272, no. 31, 01 August 1997, pages 19518 - 19524, XP002937425 *
SHARMA ET AL.: 'Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo' THE JOURNAL OF IMMUNOLOGY vol. 164, 01 May 2000, pages 4558 - 4563, XP002953294 *
SOTO ET AL.: 'The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3' PROC. NATL. ACAD. SCI. USA vol. 95, July 1998, pages 8205 - 8210, XP002144765 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019057808A1 (fr) 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations

Also Published As

Publication number Publication date
AU2002256268A1 (en) 2002-11-05
WO2002085286A3 (fr) 2004-04-01
US20030175801A1 (en) 2003-09-18
WO2002085286A8 (fr) 2003-04-03

Similar Documents

Publication Publication Date Title
US20220162279A1 (en) Combination immunotherapy
Tatsumi et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity
Narvaiza et al. Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-γ-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy
Hess et al. Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-γ
US20040009939A1 (en) Methods of enhancing immune induction involving MDA-7
US5986059A (en) T-cell selective interleukin-4 agonists
US11786569B2 (en) Expression of metabolic modulators in tumor microenvironment to improve tumor therapy
CN100543036C (zh) 递送治疗或诊断试剂的导向蛋白质
US20160058837A1 (en) Methods of using secondary lymphoid organ chemokine to modulate physiological processes in mammals
WO2002085286A2 (fr) Procedes permettant d'utiliser la chimiokine d'organes lymphoides secondaires pour moduler des processus physiologiques chez des mammiferes
US20020183271A1 (en) Methods of treatment involving human MDA-7
US6335426B1 (en) T-cell selective interleukin-4 agonists
EP1641491B1 (fr) Infiltration accrue de lymphocytes t dans une tumeur par le mutant light
RU2235728C2 (ru) Мутеин человеческого il-4, полинуклеотидная молекула, вектор, линия sf9, трансформированная вектором, способ выбора мутеина, фармацевтическая композиция для активирования т-клеток, способ лечения пациента, страдающего заболеванием, поддающимся лечению il-4
JP2001524818A (ja) ケモカインβ−６
US20040043040A1 (en) SDF-1 beta tumor vaccines and uses therefor
WO1994019022A1 (fr) Composition pour le traitement de tumeurs et l'immunisation d'organismes a l'encontre de tumeurs
US20190183967A1 (en) Compositions and methods for treating fibrosis
CN1094974C (zh) 表达人白细胞介素12的重组腺病毒及其制法和用途
Jang et al. Administration of multiple cytokine genes with anti-tumor activity inhibits both tumor incidence and tumor growth
Kim et al. Retroviral-mediated IL-12 gene therapy for advanced murine tumors
Deisseroth Genetic systems for the diagnosis and treatment of human disease
WO2003035106A1 (fr) Procedes et compositions servant a induire une reponse immune

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP